write an essay on factors affecting enzyme activity

Microbiology Notes

Factors That Affects Enzyme Activity

Table of Contents

What is Enzyme?

• Enzymes are remarkable proteins that play a pivotal role in catalyzing non-spontaneous chemical reactions within biological systems. As vital components of an organism’s metabolic pathways, enzymes ensure the smooth execution of cellular tasks necessary for growth, repair, and energy acquisition.
• The metabolic network, a complex web of interconnected chemical reactions, adapts its activities in response to internal and external stimuli. For an organism to function and survive effectively, these metabolic responses must be specific in timing and circumstances. Regulatory enzymes are key players in this process, as they control the overall rate of metabolic pathways, determining the pace at which cellular tasks are accomplished.
• Enzymatic reactions are remarkably precise, occurring only in suitable cellular environments and proceeding at a rate proportional to the availability of required substrates or cofactors. The surrounding conditions heavily influence enzyme activity, with factors such as temperature, pH, substrate concentration, and the presence of inhibitors or activators affecting the rate of these chemical reactions.
• Biochemical reactions are the foundation of an organism’s growth, tissue repair, and energy generation. These essential reactions, collectively known as metabolism, continuously occur within all living organisms. If metabolism ceases to function, it inevitably leads to the organism’s demise.
• Metabolic reactions demand high activation energy to occur, which can be energetically costly for cells. To minimize energy consumption, nature has devised a solution – the enzyme. Enzymes serve as biological catalysts, consisting of large protein molecules that accelerate chemical reactions inside cells. Composed of chains of polypeptides made up of amino acids, enzymes remain unchanged during the reaction, enabling them to speed up reactions without being consumed.
• Enzymes exhibit a remarkable specificity compared to other catalysts, with each enzyme specialized for a particular substrate or a select few substrates, thereby ensuring the precise execution of specific reactions. By lowering the activation energy required to initiate a reaction, enzymes facilitate the rapid progress of essential cellular processes.
• Several factors influence the speed of an enzyme’s action. The concentration of the enzyme and substrate, as well as environmental factors like temperature and pH, play significant roles in determining the enzyme’s efficiency. Additionally, the presence of inhibitors can hinder the enzyme’s activity, while activators can enhance it.
• In summary, enzymes are essential and fascinating biological catalysts that orchestrate vital chemical reactions within living organisms. Through their specific and efficient actions, enzymes contribute to the overall functioning and survival of all life forms. Understanding the intricacies of enzymes not only advances our knowledge of biological systems but also opens the door to numerous applications in various fields, including medicine, biotechnology, and environmental science.

Enzyme activity, a crucial aspect of biological processes, is influenced by several key factors that dictate the rate and efficiency of enzymatic reactions. Understanding these factors is vital for comprehending the intricacies of enzyme functioning. Let us delve into the six main factors that affect enzyme activity:

• Concentration of Enzyme: The availability of enzymes significantly impacts the rate of enzymatic reactions. Higher enzyme concentrations generally lead to more rapid reactions, as there are more enzymes available to interact with substrates. Conversely, low enzyme concentrations can limit the reaction rate, even if an ample amount of substrate is present.
• Concentration of Substrate: The concentration of the substrate, the molecule upon which the enzyme acts, is another crucial determinant of enzyme activity. When substrate concentration increases, the probability of substrate-enzyme collisions rises, leading to more frequent enzyme-substrate interactions and, consequently, increased reaction rates. However, once the enzyme reaches its saturation point, further increases in substrate concentration will not enhance the reaction rate.
• Effect of Temperature : Temperature profoundly influences enzyme activity. As the temperature rises, so does the kinetic energy of molecules, including enzymes and substrates. This heightened energy results in more frequent collisions between the enzyme and substrate, thereby accelerating the reaction rate. However, excessively high temperatures can denature the enzyme, rendering it non-functional.
• Effect of pH: The pH level of the surrounding environment plays a critical role in enzyme activity. Enzymes exhibit optimal activity at specific pH values, which vary depending on the type of enzyme. Deviations from the optimal pH can denature the enzyme or alter its active site, leading to reduced catalytic efficiency.
• Effect of Product Concentration: The accumulation of reaction products can influence enzyme activity. In some cases, high product concentrations can inhibit enzyme activity through negative feedback mechanisms, ensuring that excessive product formation is controlled. This phenomenon helps maintain cellular homeostasis.
• Effect of Activators: Activators are molecules that enhance enzyme activity. These substances can bind to enzymes and induce conformational changes that increase the enzyme’s affinity for its substrate or stabilize the enzyme-substrate complex. As a result, the rate of the enzymatic reaction is boosted.

In summary, enzyme activity is subject to a delicate interplay of various factors. The concentration of both enzymes and substrates, environmental conditions such as temperature and pH, and the influence of reaction products and activators collectively govern the efficiency of enzymatic reactions. Proper regulation of these factors is essential for maintaining optimal enzyme activity, enabling organisms to carry out essential biochemical processes with precision and efficiency. Researchers continue to explore these factors to gain deeper insights into enzymatic mechanisms and develop practical applications across diverse fields, including medicine, biotechnology, and environmental science.

Factor 1: Enzyme Concentration

• Enzyme concentration is a critical factor that profoundly influences the rate and efficiency of enzymatic reactions. Enzymes play a pivotal role as catalysts, facilitating chemical reactions by forming transient bonds with their substrates, thus lowering the activation energy required for the reaction to occur. Moreover, enzymes stabilize the transition state, making the reaction more favorable.
• When the enzyme concentration is high, more enzyme molecules are available to interact with the substrates. This abundance of enzyme-substrate complexes leads to a higher initial catalytic rate, giving the reaction a headstart in progressing towards reactant-product equilibrium. In contrast, reactions with the same enzyme but at lower concentrations may take longer to achieve equilibrium.
• Enzyme concentration becomes particularly significant when the substrate concentration is abundant, and the limiting factor becomes the availability of enzymes. In such cases, a higher enzyme concentration allows for more rapid formation of enzyme-substrate complexes, leading to a faster overall reaction rate.
• However, it is important to note that enzyme concentration has its limits. Beyond a certain point, increasing the enzyme concentration will not necessarily lead to a proportional increase in the reaction rate. This is because other factors, such as substrate concentration, reaction conditions, and the availability of cofactors, also play roles in governing the overall reaction rate.
• Enzyme concentration can be regulated by the cell in response to various physiological conditions. Cells may adjust the production of enzymes through gene regulation or control their activation and deactivation to maintain the appropriate balance between enzyme and substrate concentrations.
• In summary, enzyme concentration is a critical factor that directly impacts the rate of enzymatic reactions. A higher enzyme concentration results in a larger number of available enzymes to form enzyme-substrate complexes, leading to a more rapid initial catalytic rate. Understanding and controlling enzyme concentration are essential for regulating cellular processes and ensuring that biochemical reactions occur with precision and efficiency.

Factor 2: Substrate Concentration

• Substrate concentration is a crucial factor that significantly impacts the rate and efficiency of enzymatic reactions. For enzyme catalytic activity to occur, the substrate must be geometrically and electronically complementary to the enzyme’s catalytic or active site. When a substrate binds to the active site, the enzyme’s active residues transiently interact with the substrate, catalyzing its transformation into a product.
• The relationship between enzyme activity and substrate concentration can be described by different kinetic models, depending on the type of enzyme. Most enzymes follow the Michaelis-Menten kinetics, which consists of two stages. Initially, the relationship between enzyme activity and substrate concentration shows a linear association, where the reaction rate increases with increasing substrate concentration. However, this trend eventually plateaus as the number of unbound active sites decreases, reaching a maximum rate known as the maximum velocity (Vmax).
• On the other hand, allosteric enzymes exhibit a sigmoidal kinetic pattern. In the initial stages of the reaction, the relationship between the rate of an allosteric enzyme-catalyzed reaction and substrate concentration is exponential. As catalysis progresses and substrate binding saturates the enzyme’s active sites, the rate becomes linear, eventually reaching a plateau.
• In the presence of a fixed amount of enzyme, the rate of an enzymatic reaction initially increases with increasing substrate concentration until it reaches a limiting rate. At this point, the enzyme’s active sites become fully occupied by substrate molecules, and the enzyme is said to be saturated with substrate. Any further increase in substrate concentration does not significantly affect the reaction rate because the excess substrate molecules cannot react until the substrate already bound to the enzymes has undergone the reaction and been released.
• This phenomenon highlights the importance of proper substrate regulation in enzymatic reactions. Cells carefully control substrate concentrations to maintain the optimal efficiency of enzymatic processes. Substrate concentration is influenced by factors such as substrate availability, cellular demand, and the presence of regulatory molecules.
• In conclusion, substrate concentration plays a fundamental role in enzymatic reactions. The availability of substrate molecules dictates the rate at which enzyme-substrate complexes form and, subsequently, the overall reaction rate. Proper understanding and regulation of substrate concentration are essential for cells to maintain precise control over biochemical processes and ensure efficient utilization of resources.

Factor 3: Effect of Temperature

• The effect of temperature on enzyme activity is a critical factor that significantly influences the efficiency of enzymatic reactions. Like pH, temperature plays a crucial role in the stability of an enzyme’s intramolecular bonds, which in turn affects its overall activity. Generally, enzymes exhibit optimal activity at their specific optimal temperature.
• A slight increase in temperature can accelerate the reaction rate as the reactants gain more kinetic energy, leading to more frequent collisions between enzymes and substrates. This results in an increase in the rate of enzyme-substrate interactions and, consequently, a faster reaction rate. However, significant deviations from the optimal temperature can have adverse effects on enzyme activity.
• At extremely high temperatures, the intramolecular bonds and enzyme conformation can be disrupted, leading to permanent denaturation and rendering the enzyme non-functional. This phenomenon is commonly observed in extreme thermophiles, such as Thermococcus hydrothermalis and Sulfolobus solfataricus, which thrive in high-temperature environments and possess enzymes adapted to such conditions.
• Conversely, at low temperatures, the kinetic energy of the system decreases, leading to reduced reaction rates. Enzyme activity declines as the temperature falls below the optimal range. Unlike high temperatures, low temperatures do not necessarily cause permanent enzyme denaturation, and enzyme activity may be restored once the temperature returns to the optimal range.
• However, excessively low temperatures can lead to decreased solubility of enzymes in aqueous solutions, causing them to unfold and become inactive. Furthermore, freezing and thawing processes can damage enzymes irreversibly, particularly when ice crystals form and disrupt the enzyme’s protein structure. To minimize freeze-thaw damage, it is essential to minimize freeze-thaw cycles, control freezing or thawing durations, and use additives like sucrose or glycerol to protect the enzyme during freezing.
• Enzymes’ protein nature makes them extremely sensitive to thermal changes, and their activity is restricted to a narrow range of temperatures compared to ordinary chemical reactions. Each enzyme has an optimal temperature, typically ranging from 37 to 40°C. Beyond this range, enzyme activity gradually decreases until it reaches a temperature at which the enzyme becomes completely inactive due to alterations in its natural composition.
• In summary, temperature is a significant factor affecting enzyme activity. Enzymes exhibit optimal activity at specific temperatures, and deviations from this range can lead to reduced enzymatic efficiency or permanent denaturation. Proper temperature regulation is crucial for maintaining optimal enzyme activity and ensuring the precise and efficient execution of biochemical reactions in living organisms.

Factor 4: Effect of pH

• The effect of pH on enzyme activity is a critical factor that significantly influences the efficiency of enzymatic reactions. Enzymes, being protein molecules, consist of a chain of amino acids with electrical charges derived from the sequence of their residues. The specific arrangement of these amino acids determines the enzyme’s three-dimensional structure and its functional residues, including those found at the active site.
• Enzymes exhibit a remarkable degree of substrate specificity, allowing them to function optimally within a narrow range of pH. Most enzymes perform at their best in slightly acidic or basic pH conditions. However, some enzymes are naturally adapted to extreme acidic or basic environments, and they exhibit maximum activity within these specific pH ranges.
• A change in the pH value, whether it becomes more acidic or basic, affects the ionization of amino acid residues within the enzyme. This, in turn, leads to alterations in the three-dimensional structure of the enzyme, which directly impacts its interaction with substrates and, consequently, reduces its overall activity.
• Another significant effect of pH changes is on the enzyme’s catalytic capability. Acid-base and covalent catalysis mechanisms can be hindered or suppressed by shifts in pH, thereby influencing the enzyme’s ability to catalyze reactions effectively. In extreme cases, substantial pH changes can lead to the denaturation of the enzyme, disrupting its three-dimensional structure and rendering it permanently non-functional.
• The pH scale is used to measure the concentration of hydrogen ions (H+) in a solution, determining whether a liquid is acidic, basic, or neutral. A pH below 7 is considered acidic, while a pH above 7 is alkaline or basic. A pH of 7 represents neutrality, similar to the acidity of pure water at 25°C. Enzymes are particularly affected by changes in pH due to the presence of acidic carboxylic groups (COOH-) and basic amino groups (NH2) in their structure.
• Each enzyme has an optimal pH value at which it operates with maximum efficiency. Deviations from this optimal pH result in a decrease in enzyme activity until it eventually stops working. For example, pepsin, a digestive enzyme, operates optimally at low pH levels (high acidity), while trypsin, another digestive enzyme, functions best at high pH levels (basic conditions). Many enzymes work optimally at a neutral pH of 7.4, reflecting the physiological pH of the human body.
• In conclusion, pH is a critical factor influencing enzyme activity due to its impact on the enzyme’s three-dimensional structure, substrate specificity, and catalytic capability. Proper regulation of pH is essential for maintaining the optimal activity of enzymes in different cellular environments, ensuring the precise and efficient execution of biochemical reactions necessary for the functioning and survival of living organisms.

Factor 5: Effect of Effector or Inhibitor

• The effect of effectors or inhibitors is a critical factor that regulates the catalytic function of many enzymes. Enzymes often require the presence of non-substrate and non-enzyme molecules to initiate or modulate their activity. For instance, certain enzymes rely on metal ions or cofactors to establish their catalytic activity, while others depend on effectors to regulate their catalytic function, and this is especially evident in allosteric enzymes.
• Effectors play a vital role in activating or inhibiting enzyme activity, promoting or preventing their binding to substrates. Allosteric enzymes are a prime example, where effectors can bind to regulatory sites away from the active site, inducing conformational changes that either enhance or hinder the enzyme’s catalytic activity. This allows cells to fine-tune enzyme function in response to changing metabolic demands or external signals.
• On the other hand, inhibitors can bind to enzymes or their substrates, interfering with the ongoing enzymatic activity and preventing further catalytic events. The impact of inhibitors on enzyme activity can be either reversible or irreversible. Reversible inhibitors only render the enzyme inactive when bound to it and can be displaced by other molecules. In contrast, irreversible inhibitors form strong bonds with the enzyme’s functional groups, leading to permanent inactivation of the enzyme.
• Competitive inhibitors are a type of reversible inhibitor that competes with substrates for binding to the enzyme’s catalytic sites. By occupying the active site, they prevent the substrate from accessing it, reducing the enzyme’s catalytic activity. Non-competitive inhibitors, another type of reversible inhibitor, do not bind to the active site but instead bind to an allosteric site, leading to conformational changes that decrease the enzyme’s activity.
• Uncompetitive inhibitors are another form of reversible inhibition that binds only to the enzyme-substrate complex. By doing so, they prevent the release of the product, leading to a reduction in the overall reaction rate.
• Understanding the role of effectors and inhibitors is essential for regulating enzyme activity and maintaining the delicate balance of biochemical processes within cells. The presence or absence of specific effectors or inhibitors can modulate enzyme activity and influence cellular functions in various physiological and pathological conditions.
• Moreover, enzymes often rely on certain inorganic metallic cations, such as Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, and K+, for their optimal activity. In some cases, rare anions, like chloride ions (CI-), are also needed for specific enzyme activities, such as amylase.
• In summary, the effect of effectors or inhibitors is a critical regulatory factor that governs enzyme activity. These non-substrate and non-enzyme molecules can either activate or inhibit enzyme function, ensuring precise control over biochemical reactions in response to cellular demands and external stimuli. Understanding the interplay between effectors, inhibitors, and cofactors is fundamental to advancing our knowledge of enzyme regulation and developing potential therapeutic interventions for various medical conditions.

Factor 6: Effect of Product Concentration

• The effect of product concentration is a critical factor that can influence the velocity and efficiency of enzymatic reactions. As enzymes catalyze reactions, they convert substrates into products. The accumulation of reaction products can have a significant impact on enzyme activity.
• In some cases, the reaction products may bind to the active site of the enzyme, forming a loose complex. This product-enzyme complex can inhibit the enzyme’s activity, reducing its catalytic efficiency. This type of inhibition is known as product inhibition.
• Product inhibition is an important regulatory mechanism in living systems. In order to prevent the negative effects of product inhibition, living organisms have evolved mechanisms to rapidly remove or utilize the products formed during enzymatic reactions. This quick removal of products prevents the accumulation of inhibitory molecules at the enzyme’s active site, allowing the enzyme to continue functioning effectively.
• There are various ways in which living systems prevent or mitigate the effects of product inhibition. One common strategy is the presence of specific enzymes dedicated to the degradation or utilization of the reaction products. These secondary enzymes act on the products, converting them into other substances that can be further utilized or excreted from the cell.
• Additionally, regulatory mechanisms, such as feedback inhibition, play a role in preventing excessive product accumulation. Feedback inhibition occurs when the final product of a metabolic pathway acts as an inhibitor of one of the earlier enzymes in the pathway. This creates a negative feedback loop that regulates the rate of the entire pathway, preventing the overproduction of the final product.
• Overall, the effect of product concentration on enzyme activity is a crucial consideration in cellular processes. Product inhibition can slow down enzymatic reactions and potentially disrupt metabolic pathways. However, living systems have evolved sophisticated mechanisms to mitigate the effects of product inhibition, ensuring that enzymatic reactions proceed with precision and efficiency, and maintaining the delicate balance necessary for proper cellular functioning.
• Factor 1: Enzyme Concentration Example: Imagine a scenario where you have an enzyme that breaks down a complex sugar molecule into simpler sugars. If you increase the concentration of this enzyme in a test tube containing the substrate (complex sugar), the reaction rate will increase as more enzyme molecules are available to interact with the substrate. Conversely, if you decrease the enzyme concentration, the reaction rate will slow down due to a lower number of available enzymes to catalyze the reaction.
• Factor 2: Substrate Concentration Example: Let’s consider an enzyme that converts hydrogen peroxide (H2O2) into water (H2O) and oxygen gas (O2). If you increase the concentration of hydrogen peroxide in the reaction mixture, the enzyme’s activity will increase, as there will be more substrate molecules available for the enzyme to act upon. However, once all the enzyme’s active sites are fully occupied with substrate molecules, further increases in hydrogen peroxide concentration will not affect the reaction rate.
• Factor 3: Effect of Temperature Example: Take the example of an enzyme that functions optimally at body temperature (around 37°C). At this temperature, the enzyme’s activity is at its highest because the kinetic energy of molecules is sufficient for efficient enzyme-substrate collisions. However, if you increase the temperature beyond a certain point, such as 50°C, the enzyme’s three-dimensional structure may start to denature, leading to a significant reduction in enzyme activity.
• Factor 4: Effect of pH Example: Consider an enzyme responsible for breaking down proteins in the stomach. This enzyme, known as pepsin, works optimally at a low pH (acidic environment) of around 2.0. If you change the pH to a more basic environment (higher pH), pepsin’s activity will decrease significantly because the enzyme’s active site is adapted to function best in an acidic environment.
• Factor 5: Effect of Activators Example: An example of an enzyme that requires an activator is the enzyme hexokinase, which catalyzes the first step in glucose metabolism. Hexokinase requires the presence of magnesium ions (Mg2+) as a cofactor to function properly. The magnesium ions serve as activators, enhancing the enzyme’s catalytic activity and facilitating the conversion of glucose to glucose-6-phosphate.
• Factor 6: Effect of Product Concentration Example: Imagine an enzyme that converts a precursor molecule (A) into a product (B). As the enzyme catalyzes the reaction, the concentration of product B starts to increase. If the product concentration becomes too high, B molecules may start to bind to the enzyme’s active site, leading to product inhibition. This inhibitory effect slows down the enzyme’s activity, preventing the excessive accumulation of product B. The cell may have additional enzymes or regulatory mechanisms to utilize or remove product B from the reaction to maintain enzyme activity.

What factors affect enzyme activity?

Enzyme activity can be influenced by several factors, including temperature, pH, enzyme concentration, substrate concentration, presence of activators, and the concentration of reaction products.

How does temperature affect enzyme activity?

Temperature can significantly impact enzyme activity. Enzymes generally have an optimal temperature at which they work most efficiently. Deviations from this optimal temperature can either increase or decrease enzyme activity, with extreme temperatures leading to denaturation and loss of function.

How does pH affect enzyme activity?

The pH level of the environment in which enzymes operate can affect their activity. Each enzyme has an optimal pH at which it functions best. Deviations from this pH can alter the enzyme’s conformation and reduce its catalytic efficiency.

What is the relationship between enzyme concentration and activity?

Higher enzyme concentrations usually result in faster enzymatic reactions, as more enzyme molecules are available to interact with substrates. However, beyond a certain point, increasing enzyme concentration may not lead to a proportional increase in reaction rate.

How does substrate concentration influence enzyme activity?

Increasing substrate concentration generally increases enzyme activity until a saturation point is reached. At this point, all enzyme active sites are occupied by substrates, and further increases in substrate concentration do not significantly affect the reaction rate.

What are enzyme activators?

Enzyme activators are molecules that enhance the catalytic activity of enzymes. They may bind to the enzyme and induce conformational changes that increase the enzyme’s affinity for its substrate, leading to more efficient reactions.

How do inhibitors affect enzyme activity?

Inhibitors are molecules that reduce or completely halt enzyme activity. They can either bind to the active site of the enzyme (competitive inhibition) or to an allosteric site, leading to conformational changes that hinder catalytic activity (non-competitive inhibition).

What is product inhibition?

Product inhibition occurs when the accumulation of reaction products inhibits the enzyme’s activity. The products can bind to the enzyme’s active site or allosteric sites, reducing its ability to catalyze further reactions.

How do living systems prevent product inhibition?

Living systems have evolved mechanisms to prevent or mitigate product inhibition. These mechanisms include rapid removal of products, utilization of secondary enzymes to degrade or utilize products, and feedback inhibition, where the final product of a metabolic pathway regulates an earlier enzyme in the pathway.

Why is enzyme activity regulation important?

Regulation of enzyme activity is crucial for maintaining proper metabolic balance and cellular function. Precise control of enzymatic reactions ensures that biochemical processes occur at the right time, in the right place, and with the appropriate rate to meet the demands of the organism.

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Enzymes: principles and biotechnological applications

Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed.

The nature and classification of enzymes

Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms. They can also be extracted from cells and then used to catalyse a wide range of commercially important processes. For example, they have important roles in the production of sweetening agents and the modification of antibiotics, they are used in washing powders and various cleaning products, and they play a key role in analytical devices and assays that have clinical, forensic and environmental applications. The word ‘enzyme’ was first used by the German physiologist Wilhelm Kühne in 1878, when he was describing the ability of yeast to produce alcohol from sugars, and it is derived from the Greek words en (meaning ‘within’) and zume (meaning ‘yeast’).

In the late nineteenth century and early twentieth century, significant advances were made in the extraction, characterization and commercial exploitation of many enzymes, but it was not until the 1920s that enzymes were crystallized, revealing that catalytic activity is associated with protein molecules. For the next 60 years or so it was believed that all enzymes were proteins, but in the 1980s it was found that some ribonucleic acid (RNA) molecules are also able to exert catalytic effects. These RNAs, which are called ribozymes, play an important role in gene expression. In the same decade, biochemists also developed the technology to generate antibodies that possess catalytic properties. These so-called ‘abzymes’ have significant potential both as novel industrial catalysts and in therapeutics. Notwithstanding these notable exceptions, much of classical enzymology, and the remainder of this essay, is focused on the proteins that possess catalytic activity.

As catalysts, enzymes are only required in very low concentrations, and they speed up reactions without themselves being consumed during the reaction. We usually describe enzymes as being capable of catalysing the conversion of substrate molecules into product molecules as follows:

Enzymes are potent catalysts

The enormous catalytic activity of enzymes can perhaps best be expressed by a constant, k cat , that is variously referred to as the turnover rate, turnover frequency or turnover number. This constant represents the number of substrate molecules that can be converted to product by a single enzyme molecule per unit time (usually per minute or per second). Examples of turnover rate values are listed in Table 1 . For example, a single molecule of carbonic anhydrase can catalyse the conversion of over half a million molecules of its substrates, carbon dioxide (CO 2 ) and water (H 2 O), into the product, bicarbonate (HCO 3 − ), every second—a truly remarkable achievement.

Enzymes are specific catalysts

As well as being highly potent catalysts, enzymes also possess remarkable specificity in that they generally catalyse the conversion of only one type (or at most a range of similar types) of substrate molecule into product molecules.

Some enzymes demonstrate group specificity. For example, alkaline phosphatase (an enzyme that is commonly encountered in first-year laboratory sessions on enzyme kinetics) can remove a phosphate group from a variety of substrates.

Other enzymes demonstrate much higher specificity, which is described as absolute specificity. For example, glucose oxidase shows almost total specificity for its substrate, β-D-glucose, and virtually no activity with any other monosaccharides. As we shall see later, this specificity is of paramount importance in many analytical assays and devices (biosensors) that measure a specific substrate (e.g. glucose) in a complex mixture (e.g. a blood or urine sample).

Enzyme names and classification

Enzymes typically have common names (often called ‘trivial names’) which refer to the reaction that they catalyse, with the suffix -ase (e.g. oxidase, dehydrogenase, carboxylase), although individual proteolytic enzymes generally have the suffix - in (e.g. trypsin, chymotrypsin, papain). Often the trivial name also indicates the substrate on which the enzyme acts (e.g. glucose oxidase, alcohol dehydrogenase, pyruvate decarboxylase). However, some trivial names (e.g. invertase, diastase, catalase) provide little information about the substrate, the product or the reaction involved.

Due to the growing complexity of and inconsistency in the naming of enzymes, the International Union of Biochemistry set up the Enzyme Commission to address this issue. The first Enzyme Commission Report was published in 1961, and provided a systematic approach to the naming of enzymes. The sixth edition, published in 1992, contained details of nearly 3 200 different enzymes, and supplements published annually have now extended this number to over 5 000.

Within this system, all enzymes are described by a four-part Enzyme Commission (EC) number. For example, the enzyme with the trivial name lactate dehydrogenase has the EC number 1.1.1.27, and is more correctly called l –lactate: NAD + oxidoreductase.

The first part of the EC number refers to the reaction that the enzyme catalyses ( Table 2 ). The remaining digits have different meanings according to the nature of the reaction identified by the first digit. For example, within the oxidoreductase category, the second digit denotes the hydrogen donor ( Table 3 ) and the third digit denotes the hydrogen acceptor ( Table 4 ).

Thus lactate dehydrogenase with the EC number 1.1.1.27 is an oxidoreductase (indicated by the first digit) with the alcohol group of the lactate molecule as the hydrogen donor (second digit) and NAD + as the hydrogen acceptor (third digit), and is the 27th enzyme to be categorized within this group (fourth digit).

Fortunately, it is now very easy to find this information for any individual enzyme using the Enzyme Nomenclature Database (available at http://enzyme.expasy.org ).

Enzyme structure and substrate binding

Amino acid-based enzymes are globular proteins that range in size from less than 100 to more than 2 000 amino acid residues. These amino acids can be arranged as one or more polypeptide chains that are folded and bent to form a specific three-dimensional structure, incorporating a small area known as the active site ( Figure 1 ), where the substrate actually binds. The active site may well involve only a small number (less than 10) of the constituent amino acids.

It is the shape and charge properties of the active site that enable it to bind to a single type of substrate molecule, so that the enzyme is able to demonstrate considerable specificity in its catalytic activity.

The hypothesis that enzyme specificity results from the complementary nature of the substrate and its active site was first proposed by the German chemist Emil Fischer in 1894, and became known as Fischer's ‘lock and key hypothesis’, whereby only a key of the correct size and shape (the substrate) fits into the keyhole (the active site) of the lock (the enzyme). It is astounding that this theory was proposed at a time when it was not even established that enzymes were proteins. As more was learned about enzyme structure through techniques such as X-ray crystallography, it became clear that enzymes are not rigid structures, but are in fact quite flexible in shape. In the light of this finding, in 1958 Daniel Koshland extended Fischer's ideas and presented the ‘induced-fit model’ of substrate and enzyme binding, in which the enzyme molecule changes its shape slightly to accommodate the binding of the substrate. The analogy that is commonly used is the ‘hand-in-glove model’, where the hand and glove are broadly complementary in shape, but the glove is moulded around the hand as it is inserted in order to provide a perfect match.

Since it is the active site alone that binds to the substrate, it is logical to ask what is the role of the rest of the protein molecule. The simple answer is that it acts to stabilize the active site and provide an appropriate environment for interaction of the site with the substrate molecule. Therefore the active site cannot be separated out from the rest of the protein without loss of catalytic activity, although laboratory-based directed (or forced) evolution studies have shown that it is sometimes possible to generate smaller enzymes that do retain activity.

It should be noted that although a large number of enzymes consist solely of protein, many also contain a non-protein component, known as a cofactor, that is necessary for the enzyme's catalytic activity. A cofactor may be another organic molecule, in which case it is called a coenzyme, or it may be an inorganic molecule, typically a metal ion such as iron, manganese, cobalt, copper or zinc. A coenzyme that binds tightly and permanently to the protein is generally referred to as the prosthetic group of the enzyme.

When an enzyme requires a cofactor for its activity, the inactive protein component is generally referred to as an apoenzyme, and the apoenzyme plus the cofactor (i.e. the active enzyme) is called a holoenzyme ( Figure 2 ).

The need for minerals and vitamins in the human diet is partly attributable to their roles within metabolism as cofactors and coenzymes.

Enzymes and reaction equilibrium

How do enzymes work? The broad answer to this question is that they do not alter the equilibrium (i.e. the thermodynamics) of a reaction. This is because enzymes do not fundamentally change the structure and energetics of the products and reagents, but rather they simply allow the reaction equilibrium to be attained more rapidly. Let us therefore begin by clarifying the concept of chemical equilibrium.

In many cases the equilibrium of a reaction is far ‘to the right’—that is, virtually all of the substrate (S) is converted into product (P). For this reason, reactions are often written as follows:

This is a simplification, as in all cases it is more correct to write this reaction as follows:

This indicates the presence of an equilibrium. To understand this concept it is perhaps most helpful to look at a reaction where the equilibrium point is quite central.

For example:

In this reaction, if we start with a solution of 1 mol l −1 glucose and add the enzyme, then upon completion we will have a mixture of approximately 0.5 mol l −1 glucose and 0.5 mol l −1 fructose. This is the equilibrium point of this particular reaction, and although it may only take a couple of seconds to reach this end point with the enzyme present, we would in fact come to the same point if we put glucose into solution and waited many months for the reaction to occur in the absence of the enzyme. Interestingly, we could also have started this reaction with a 1 mol l −1 fructose solution, and it would have proceeded in the opposite direction until the same equilibrium point had been reached.

The equilibrium point for this reaction is expressed by the equilibrium constant K eq as follows:

Thus for a reaction with central equilibrium, K eq = 1, for an equilibrium ‘to the right’ K eq is >1, and for an equilibrium ‘to the left’ K eq is <1.

Therefore if a reaction has a K eq value of 10 6 , the equilibrium is very far to the right and can be simplified by denoting it as a single arrow. We may often describe this type of reaction as ‘going to completion’. Conversely, if a reaction has a K eq value of 10 −6 , the equilibrium is very far to the left, and for all practical purposes it would not really be considered to proceed at all.

It should be noted that although the concentration of reactants has no effect on the equilibrium point, environmental factors such as pH and temperature can and do affect the position of the equilibrium.

It should also be noted that any biochemical reaction which occurs in vivo in a living system does not occur in isolation, but as part of a metabolic pathway, which makes it more difficult to conceptualize the relationship between reactants and reactions. In vivo reactions are not allowed to proceed to their equilibrium position. If they did, the reaction would essentially stop (i.e. the forward and reverse reactions would balance each other), and there would be no net flux through the pathway. However, in many complex biochemical pathways some of the individual reaction steps are close to equilibrium, whereas others are far from equilibrium, the latter (catalysed by regulatory enzymes) having the greatest capacity to control the overall flux of materials through the pathway.

Enzymes form complexes with their substrates

We often describe an enzyme-catalysed reaction as proceeding through three stages as follows:

The ES complex represents a position where the substrate (S) is bound to the enzyme (E) such that the reaction (whatever it might be) is made more favourable. As soon as the reaction has occurred, the product molecule (P) dissociates from the enzyme, which is then free to bind to another substrate molecule. At some point during this process the substrate is converted into an intermediate form (often called the transition state) and then into the product.

The exact mechanism whereby the enzyme acts to increase the rate of the reaction differs from one system to another. However, the general principle is that by binding of the substrate to the enzyme, the reaction involving the substrate is made more favourable by lowering the activation energy of the reaction.

In terms of energetics, reactions can be either exergonic (releasing energy) or endergonic (consuming energy). However, even in an exergonic reaction a small amount of energy, termed the activation energy, is needed to give the reaction a ‘kick start.’ A good analogy is that of a match, the head of which contains a mixture of energy-rich chemicals (phosphorus sesquisulfide and potassium chlorate). When a match burns it releases substantial amounts of light and heat energy (exergonically reacting with O 2 in the air). However, and perhaps fortunately, a match will not spontaneously ignite, but rather a small input of energy in the form of heat generated through friction (i.e. striking of the match) is needed to initiate the reaction. Of course once the match has been struck the amount of energy released is considerable, and greatly exceeds the small energy input during the striking process.

As shown in Figure 3 , enzymes are considered to lower the activation energy of a system by making it energetically easier for the transition state to form. In the presence of an enzyme catalyst, the formation of the transition state is energetically more favourable (i.e. it requires less energy for the ‘kick start’), thereby accelerating the rate at which the reaction will proceed, but not fundamentally changing the energy levels of either the reactant or the product.

Properties and mechanisms of enzyme action

Enzyme kinetics.

Enzyme kinetics is the study of factors that determine the speed of enzyme-catalysed reactions. It utilizes some mathematical equations that can be confusing to students when they first encounter them. However, the theory of kinetics is both logical and simple, and it is essential to develop an understanding of this subject in order to be able to appreciate the role of enzymes both in metabolism and in biotechnology.

Assays (measurements) of enzyme activity can be performed in either a discontinuous or continuous fashion. Discontinuous methods involve mixing the substrate and enzyme together and measuring the product formed after a set period of time, so these methods are generally easy and quick to perform. In general we would use such discontinuous assays when we know little about the system (and are making preliminary investigations), or alternatively when we know a great deal about the system and are certain that the time interval we are choosing is appropriate.

In continuous enzyme assays we would generally study the rate of an enzyme-catalysed reaction by mixing the enzyme with the substrate and continuously measuring the appearance of product over time. Of course we could equally well measure the rate of the reaction by measuring the disappearance of substrate over time. Apart from the actual direction (one increasing and one decreasing), the two values would be identical. In enzyme kinetics experiments, for convenience we very often use an artificial substrate called a chromogen that yields a brightly coloured product, making the reaction easy to follow using a colorimeter or a spectrophotometer. However, we could in fact use any available analytical equipment that has the capacity to measure the concentration of either the product or the substrate.

In almost all cases we would also add a buffer solution to the mixture. As we shall see, enzyme activity is strongly influenced by pH, so it is important to set the pH at a specific value and keep it constant throughout the experiment.

Our first enzyme kinetics experiment may therefore involve mixing a substrate solution (chromogen) with a buffer solution and adding the enzyme. This mixture would then be placed in a spectrophotometer and the appearance of the coloured product would be measured. This would enable us to follow a rapid reaction which, after a few seconds or minutes, might start to slow down, as shown in Figure 4 .

A common reason for this slowing down of the speed (rate) of the reaction is that the substrate within the mixture is being used up and thus becoming limiting. Alternatively, it may be that the enzyme is unstable and is denaturing over the course of the experiment, or it could be that the pH of the mixture is changing, as many reactions either consume or release protons. For these reasons, when we are asked to specify the rate of a reaction we do so early on, as soon as the enzyme has been added, and when none of the above-mentioned limitations apply. We refer to this initial rapid rate as the initial velocity ( v 0 ). Measurement of the reaction rate at this early stage is also quite straightforward, as the rate is effectively linear, so we can simply draw a straight line and measure the gradient (by dividing the concentration change by the time interval) in order to evaluate the reaction rate over this period.

We may now perform a range of similar enzyme assays to evaluate how the initial velocity changes when the substrate or enzyme concentration is altered, or when the pH is changed. These studies will help us to characterize the properties of the enzyme under study.

The relationship between enzyme concentration and the rate of the reaction is usually a simple one. If we repeat the experiment just described, but add 10% more enzyme, the reaction will be 10% faster, and if we double the enzyme concentration the reaction will proceed twice as fast. Thus there is a simple linear relationship between the reaction rate and the amount of enzyme available to catalyse the reaction ( Figure 5 ).

This relationship applies both to enzymes in vivo and to those used in biotechnological applications, where regulation of the amount of enzyme present may control reaction rates.

When we perform a series of enzyme assays using the same enzyme concentration, but with a range of different substrate concentrations, a slightly more complex relationship emerges, as shown in Figure 6 . Initially, when the substrate concentration is increased, the rate of reaction increases considerably. However, as the substrate concentration is increased further the effects on the reaction rate start to decline, until a stage is reached where increasing the substrate concentration has little further effect on the reaction rate. At this point the enzyme is considered to be coming close to saturation with substrate, and demonstrating its maximal velocity ( V max ). Note that this maximal velocity is in fact a theoretical limit that will not be truly achieved in any experiment, although we might come very close to it.

The relationship described here is a fairly common one, which a mathematician would immediately identify as a rectangular hyperbola. The equation that describes such a relationship is as follows:

The two constants a and b thus allow us to describe this hyperbolic relationship, just as with a linear relationship ( y = mx + c ), which can be expressed by the two constants m (the slope) and c (the intercept).

We have in fact already defined the constant a — it is V max . The constant b is a little more complex, as it is the value on the x -axis that gives half of the maximal value of y . In enzymology we refer to this as the Michaelis constant ( K m ), which is defined as the substrate concentration that gives half-maximal velocity.

Our final equation, usually called the Michaelis–Menten equation, therefore becomes:

In 1913, Leonor Michaelis and Maud Menten first showed that it was in fact possible to derive this equation mathematically from first principles, with some simple assumptions about the way in which an enzyme reacts with a substrate to form a product. Central to their derivation is the concept that the reaction takes place via the formation of an ES complex which, once formed, can either dissociate (productively) to release product, or else dissociate in the reverse direction without any formation of product. Thus the reaction can be represented as follows, with k 1 , k −1 and k 2 being the rate constants of the three individual reaction steps:

The Michaelis–Menten derivation requires two important assumptions. The first assumption is that we are considering the initial velocity of the reaction ( v 0 ), when the product concentration will be negligibly small (i.e. [S] ≫ [P]), such that we can ignore the possibility of any product reverting to substrate. The second assumption is that the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S]≫[E]).

The derivation begins with an equation for the expression of the initial rate, the rate of formation of product, as the rate at which the ES complex dissociates to form product. This is based upon the rate constant k 2 and the concentration of the ES complex, as follows:

Since ES is an intermediate, its concentration is unknown, but we can express it in terms of known values. In a steady-state approximation we can assume that although the concentration of substrate and product changes, the concentration of the ES complex itself remains constant. The rate of formation of the ES complex and the rate of its breakdown must therefore balance, where:

Hence, at steady state:

This equation can be rearranged to yield [ES] as follows:

The Michaelis constant K m can be defined as follows:

Equation 2 may thus be simplified to:

Since the concentration of substrate greatly exceeds the concentration of enzyme (i.e. [S] ≫ [E]), the concentration of uncombined substrate [S] is almost equal to the total concentration of substrate. The concentration of uncombined enzyme [E] is equal to the total enzyme concentration [E] T minus that combined with substrate [ES]. Introducing these terms to Equation 3 and solving for ES gives us the following:

We can then introduce this term into Equation 1 to give:

The term k 2 [E] T in fact represents V max , the maximal velocity. Thus Michaelis and Menten were able to derive their final equation as:

A more detailed derivation of the Michaelis–Menten equation can be found in many biochemistry textbooks (see section 4 of Recommended Reading section). There are also some very helpful web-based tutorials available on the subject.

Michaelis constants have been determined for many commonly used enzymes, and are typically in the lower millimolar range ( Table 5 ).

It should be noted that enzymes which catalyse the same reaction, but which are derived from different organisms, can have widely differing K m values. Furthermore, an enzyme with multiple substrates can have quite different K m values for each substrate.

A low K m value indicates that the enzyme requires only a small amount of substrate in order to become saturated. Therefore the maximum velocity is reached at relatively low substrate concentrations. A high K m value indicates the need for high substrate concentrations in order to achieve maximum reaction velocity. Thus we generally refer to K m as a measure of the affinity of the enzyme for its substrate—in fact it is an inverse measure, where a high K m indicates a low affinity, and vice versa.

The K m value tells us several important things about a particular enzyme.

• An enzyme with a low K m value relative to the physiological concentration of substrate will probably always be saturated with substrate, and will therefore act at a constant rate, regardless of variations in the concentration of substrate within the physiological range.
• An enzyme with a high K m value relative to the physiological concentration of substrate will not be saturated with substrate, and its activity will therefore vary according to the concentration of substrate, so the rate of formation of product will depend on the availability of substrate.
• If an enzyme acts on several substrates, the substrate with the lowest K m value is frequently assumed to be that enzyme's ‘natural’ substrate, although this may not be true in all cases.
• If two enzymes (with similar V max ) in different metabolic pathways compete for the same substrate, then if we know the K m values for the two enzymes we can predict the relative activity of the two pathways. Essentially the pathway that has the enzyme with the lower K m value is likely to be the ‘preferred pathway’, and more substrate will flow through that pathway under most conditions. For example, phosphofructokinase (PFK) is the enzyme that catalyses the first committed step in the glycolytic pathway, which generates energy in the form of ATP for the cell, whereas glucose-1-phosphate uridylyltransferase (GUT) is an enzyme early in the pathway leading to the synthesis of glycogen (an energy storage molecule). Both enzymes use hexose monophosphates as substrates, but the K m of PFK for its substrate is lower than that of GUT for its substrate. Thus at lower cellular hexose phosphate concentrations, PFK will be active and GUT will be largely inactive. At higher hexose phosphate concentrations both pathways will be active. This means that the cells only store glycogen in times of plenty, and always give preference to the pathway of ATP production, which is the more essential function.

Very often it is not possible to estimate K m values from a direct plot of velocity against substrate concentration (as shown in Figure 6 ) because we have not used high enough substrate concentrations to come even close to estimating maximal velocity, and therefore we cannot evaluate half-maximal velocity and thus K m . Fortunately, we can plot our experimental data in a slightly different way in order to obtain these values. The most commonly used alternative is the Lineweaver–Burk plot (often called the double-reciprocal plot). This plot linearizes the hyperbolic curved relationship, and the line produced is easy to extrapolate, allowing evaluation of V max and K m . For example, if we obtained only the first seven data points in Figure 6 , we would have difficulty estimating V max from a direct plot as shown in Figure 7 a.

However, as shown in Figure 7 b, if these seven points are plotted on a graph of 1/velocity against 1/substrate concentration (i.e. a double-reciprocal plot), the data are linearized, and the line can be easily extrapolated to the left to provide intercepts on both the y -axis and the x -axis, from which V max and K m , respectively, can be evaluated.

One significant practical drawback of using the Lineweaver–Burk plot is the excessive influence that it gives to measurements made at the lowest substrate concentrations. These concentrations might well be the most prone to error (due to difficulties in making multiple dilutions), and result in reaction rates that, because they are slow, might also be most prone to measurement error. Often, as shown in Figure 8 , such points when transformed on the Lineweaver–Burk plot have a significant impact on the line of best fit estimated from the data, and therefore on the extrapolated values of both V max and K m . The two sets of points shown in Figure 8 are identical except for the single point at the top right, which reflects (because of the plot's double-reciprocal nature) a single point derived from a very low substrate concentration and a low reaction rate. However, this single point can have an enormous impact on the line of best fit and the accompanying estimates of kinetic constants.

In fact there are other kinetic plots that can be used, including the Eadie–Hofstee plot, the Hanes plot and the Eisenthal–Cornish-Bowden plot, which are less prone to such problems. However, the Lineweaver–Burk plot is still the most commonly described kinetic plot in the majority of enzymology textbooks, and thus retains its influence in undergraduate education.

Enzymes are affected by pH and temperature

Various environmental factors are able to affect the rate of enzyme-catalysed reactions through reversible or irreversible changes in the protein structure. The effects of pH and temperature are generally well understood.

Most enzymes have a characteristic optimum pH at which the velocity of the catalysed reaction is maximal, and above and below which the velocity declines ( Figure 9 ).

The pH profile is dependent on a number of factors. As the pH changes, the ionization of groups both at the enzyme's active site and on the substrate can alter, influencing the rate of binding of the substrate to the active site. These effects are often reversible. For example, if we take an enzyme with an optimal pH (pH opt ) of 7.0 and place it in an environment at pH 6.0 or 8.0, the charge properties of the enzyme and the substrate may be suboptimal, such that binding and hence the reaction rate are lowered. If we then readjust the pH to 7.0, the optimal charge properties and hence the maximal activity of the enzyme are often restored. However, if we place the enzyme in a more extreme acidic or alkaline environment (e.g. at pH 1 or 14), although these conditions may not actually lead to changes in the very stable covalent structure of the protein (i.e. its configuration), they may well produce changes in the conformation (shape) of the protein such that, when it is returned to pH 7.0, the original conformation and hence the enzyme's full catalytic activity are not restored.

It should be noted that the optimum pH of an enzyme may not be identical to that of its normal intracellular surroundings. This indicates that the local pH can exert a controlling influence on enzyme activity.

The effects of temperature on enzyme activity are quite complex, and can be regarded as two forces acting simultaneously but in opposite directions. As the temperature is raised, the rate of molecular movement and hence the rate of reaction increases, but at the same time there is a progressive inactivation caused by denaturation of the enzyme protein. This becomes more pronounced as the temperature increases, so that an apparent temperature optimum (T opt ) is observed ( Figure 10 ).

Thermal denaturation is time dependent, and for an enzyme the term ‘optimum temperature’ has little real meaning unless the duration of exposure to that temperature is recorded. The thermal stability of an enzyme can be determined by first exposing the protein to a range of temperatures for a fixed period of time, and subsequently measuring its activity at one favourable temperature (e.g. 25°C).

The temperature at which denaturation becomes important varies from one enzyme to another. Normally it is negligible below 30°C, and starts to become appreciable above 40°C. Typically, enzymes derived from microbial sources show much higher thermal stability than do those from mammalian sources, and enzymes derived from extremely thermophilic microorganisms, such as thermolysin (a protease from Bacillus thermoproteolyticus ) and Taq polymerase (a DNA polymerase from Thermus aquaticus ), might be completely thermostable at 70°C and still retain substantial levels of activity even at 100°C.

Enzymes are sensitive to inhibitors

Substances that reduce the activity of an enzyme-catalysed reaction are known as inhibitors. They act by either directly or indirectly influencing the catalytic properties of the active site. Inhibitors can be foreign to the cell or natural components of it. Those in the latter category can represent an important element of the regulation of cell metabolism. Many toxins and also many pharmacologically active agents (both illegal drugs and prescription and over-the-counter medicines) act by inhibiting specific enzyme-catalysed processes.

Reversible inhibition

Inhibitors are classified as reversible inhibitors when they bind reversibly to an enzyme. A molecule that is structurally similar to the normal substrate may be able to bind reversibly to the enzyme's active site and therefore act as a competitive inhibitor. For example, malonate is a competitive inhibitor of the enzyme succinate dehydrogenase, as it is capable of binding to the enzyme's active site due to its close structural similarity to the enzyme's natural substrate, succinate (see below). When malonate occupies the active site of succinate dehydrogenase it prevents the natural substrate, succinate, from binding, thereby slowing down the rate of oxidation of succinate to fumarate (i.e. inhibiting the reaction).

One of the characteristics of competitive inhibitors is that they can be displaced from the active site if high concentrations of substrate are used, thereby restoring enzyme activity. Thus competitive inhibitors increase the K m of a reaction because they increase the concentration of substrate required to saturate the enzyme. However, they do not change V max itself.

In the case of certain enzymes, high concentrations of either the substrate or the product can be inhibitory. For example, invertase activity is considerably reduced in the presence of high concentrations of sucrose (its substrate), whereas the β-galactosidase of Aspergillus niger is strongly inhibited by galactose (its product). Products of an enzyme reaction are some of the most commonly encountered competitive inhibitors.

Other types of reversible inhibitor also exist. Non-competitive inhibitors react with the enzyme at a site distinct from the active site. Therefore the binding of the inhibitor does not physically block the substrate–binding site, but it does prevent subsequent reaction. Most non-competitive inhibitors are chemically unrelated to the substrate, and their inhibition cannot be overcome by increasing the substrate concentration. Such inhibitors in effect reduce the concentration of the active enzyme in solution, thereby reducing the V max of the reaction. However, they do not change the value of K m .

Uncompetitive inhibition is rather rare, occurring when the inhibitor is only able to bind to the enzyme once a substrate molecule has itself bound. As such, inhibition is most significant at high substrate concentrations, and results in a reduction in the V max of the reaction. Uncompetitive inhibition also causes a reduction in K m , which seems somewhat counterintuitive as this means that the affinity of the enzyme for its substrate is actually increased when the inhibitor is present. This effect occurs because the binding of the inhibitor to the ES complex effectively removes ES complex and thereby affects the overall equilibrium of the reaction favouring ES complex formation. It is noteworthy however that since both V max and K m are reduced the observed reaction rates with inhibitor present are always lower than those in the absence of the uncompetitive inhibitor.

Irreversible inhibitors and poisons

If an inhibitor binds permanently to an enzyme it is known as an irreversible inhibitor. Many irreversible inhibitors are therefore potent toxins.

Organophosphorus compounds such as diisopropyl fluorophosphate (DFP) inhibit acetylcholinesterase activity by reacting covalently with an important serine residue found within the active site of the enzyme. The physiological effect of this inactivation is interference with neurotransmitter inactivation at the synapses of nerves, resulting in the constant propagation of nerve impulses, which can lead to death. DFP was originally evaluated by the British as a chemical warfare agent during World War Two, and modified versions of this compound are now widely used as organophosphate pesticides (e.g. parathione, malathione).

Allosteric regulators and the control of enzyme activity

Having spent time learning about enzyme kinetics and the Michaelis–Menten relationship, it is often quite disconcerting to find that some of the most important enzymes do not in fact display such properties. Allosteric enzymes are key regulatory enzymes that control the activities of metabolic pathways by responding to inhibitors and activators. These enzymes in fact show a sigmoidal (S-shaped) relationship between reaction rate and substrate concentration ( Figure 11 ), rather than the usual hyperbolic relationship. Thus for allosteric enzymes there is an area where activity is lower than that of an equivalent ‘normal’ enzyme, and also an area where activity is higher than that of an equivalent ‘normal’ enzyme, with a rapid transition between these two phases. This is rather like a switch that can quickly be changed from ‘off’ (low activity) to ‘on’ (full activity).

Most allosteric enzymes are polymeric—that is, they are composed of at least two (and often many more) individual polypeptide chains. They also have multiple active sites where the substrate can bind. Much of our understanding of the function of allosteric enzymes comes from studies of haemoglobin which, although it is not an enzyme, binds oxygen in a similarly co-operative way and thus also demonstrates this sigmoidal relationship. Allosteric enzymes have an initially low affinity for the substrate, but when a single substrate molecule binds, this may break some bonds within the enzyme and thereby change the shape of the protein such that the remaining active sites are able to bind with a higher affinity. Therefore allosteric enzymes are often described as moving from a tensed state or T-state (low affinity) in which no substrate is bound, to a relaxed state or R-state (high affinity) as substrate binds. Other molecules can also bind to allosteric enzymes, at additional regulatory sites (i.e. not at the active site). Molecules that stabilize the protein in its T-state therefore act as allosteric inhibitors, whereas molecules that move the protein to its R-state will act as allosteric activators or promoters.

A good example of an allosteric enzyme is aspartate transcarbamoylase (ATCase), a key regulatory enzyme that catalyses the first committed step in the sequence of reactions that produce the pyrimidine nucleotides which are essential components of DNA and RNA. The reaction is as follows:

The end product in the pathway, the pyrimidine nucleotide cytidine triphosphate (CTP), is an active allosteric inhibitor of the enzyme ATCase. Therefore when there is a high concentration of CTP in the cell, this feeds back and inhibits the ATCase enzyme, reducing its activity and thus lowering the rate of production of further pyrimidine nucleotides. As the concentration of CTP in the cell decreases then so does the inhibition of ATCase, and the resulting increase in enzyme activity leads to the production of more pyrimidine nucleotides. This negative feedback inhibition is an important element of biochemical homeostasis within the cell. However, in order to synthesize DNA and RNA, the cell requires not only pyrimidine nucleotides but also purine nucleotides, and these are needed in roughly equal proportions. Purine synthesis occurs through a different pathway, but interestingly the final product, the purine nucleotide adenosine triphosphate (ATP), is a potent activator of the enzyme ATCase. This is logical, since when the cell contains high concentrations of purine nucleotides it will require equally high concentrations of pyrimidine nucleotides in order for these two types of nucleotide to combine to form the polymers DNA and RNA. Thus ATCase is able to regulate the production of pyrimidine nucleotides within the cell according to cellular demand, and also to ensure that pyrimidine nucleotide synthesis is synchronized with purine nucleotide synthesis—an elegant biochemical mechanism for the regulation of an extremely important metabolic process.

There are some rare, although important, cases of monomeric enzymes that have only one substrate-binding site but are capable of demonstrating the sigmoidal reaction kinetics characteristic of allosteric enzymes. Particularly noteworthy in this context is the monomeric enzyme glucokinase (also called hexokinase IV), which catalyses the phosphorylation of glucose to glucose-6-phosphate (which may then either be metabolized by the glycolytic pathway or be used in glycogen synthesis). It has been postulated that this kinetic behaviour is a result of individual glucokinase molecules existing in one of two forms—a low-affinity form and a high-affinity form. The low-affinity form of the enzyme reacts with its substrate (glucose), is then turned into the high-affinity form, and remains in that state for a short time before slowly returning to its original low-affinity form (demonstrating a so-called slow transition). Therefore at high substrate concentrations the enzyme is likely to react with a second substrate molecule soon after the first one (i.e. while still in its high-affinity form), whereas at lower substrate concentrations the enzyme may transition back to its low-affinity form before it reacts with subsequent substrate molecules. This results in its characteristic sigmoidal reaction kinetics.

Origin, purification and uses of enzymes

Enzymes are ubiquitous.

Enzymes are essential components of animals, plants and microorganisms, due to the fact that they catalyse and co-ordinate the complex reactions of cellular metabolism.

Up until the 1970s, most of the commercial application of enzymes involved animal and plant sources. At that time, bulk enzymes were generally only used within the food-processing industry, and enzymes from animals and plants were preferred, as they were considered to be free from the problems of toxicity and contamination that were associated with enzymes of microbial origin. However, as demand grew and as fermentation technology developed, the competitive cost of microbial enzymes was recognized and they became more widely used.

Compared with enzymes from plant and animal sources, microbial enzymes have economic, technical and ethical advantages, which will now be outlined.

Economic advantages

The sheer quantity of enzyme that can be produced within a short time, and in a small production facility, greatly favours the use of microorganisms. For example, during the production of rennin (a milk-coagulating enzyme used in cheese manufacture) the traditional approach is to use the enzyme extracted from the stomach of a calf (a young cow still feeding on its mother's milk). The average quantity of rennet extracted from a calf's stomach is 10 kg, and it takes several months of intensive farming to produce a calf. In comparison, a 1 000-litre fermenter of recombinant Bacillus subtilis can produce 20 kg of enzyme within 12 h. Thus the microbial product is clearly preferable economically, and is free from the ethical issues that surround the use of animals. Indeed, most of the cheese now sold in supermarkets is made from milk coagulated with microbial enzymes (so is suitable for vegetarians).

A further advantage of using microbial enzymes is their ease of extraction. Many of the microbial enzymes used in biotechnological processes are secreted extracellularly, which greatly simplifies their extraction and purification. Microbial intracellular enzymes are also often easier to obtain than the equivalent animal or plant enzymes, as they generally require fewer extraction and purification steps.

Animal and plant sources usually need to be transported to the extraction facility, whereas when microorganisms are used the same facility can generally be employed for production and extraction. In addition, commercially important animal and plant enzymes are often located within only one organ or tissue, so the remaining material is essentially a waste product, disposal of which is required.

Finally, enzymes from plant and animal sources show wide variation in yield, and may only be available at certain times of year, whereas none of these problems are associated with microbial enzymes.

Technical advantages

Microbial enzymes often have properties that make them more suitable for commercial exploitation. In comparison with enzymes from animal and plant sources, the stability of microbial enzymes is usually high. For example, the high temperature stability of enzymes from thermophilic microorganisms is often useful when the process must operate at high temperatures (e.g. during starch processing).

Microorganisms are also very amenable to genetic modification to produce novel or altered enzymes, using relatively simple methods such as plasmid insertion. The genetic manipulation of animals and plants is technically much more difficult, is more expensive and is still the subject of significant ethical concern, especially in the U.K.

Enzymes may be intracellular or extracellular

Although many enzymes are retained within the cell, and may be located in specific subcellular compartments, others are released into the surrounding environment. The majority of enzymes in industrial use are extracellular proteins from either fungal sources (e.g. Aspergillus species) or bacterial sources (e.g. Bacillus species). Examples of these include α-amylase, cellulase, dextranase, proteases and amyloglucosidase. Many other enzymes for non-industrial use are intracellular and are produced in much smaller amounts by the cell. Examples of these include asparaginase, catalase, cholesterol oxidase, glucose oxidase and glucose-6-phosphate dehydrogenase.

Enzyme purification

Within the cell, enzymes are generally found along with other proteins, nucleic acids, polysaccharides and lipids. The activity of the enzyme in relation to the total protein present (i.e. the specific activity) can be determined and used as a measure of enzyme purity. A variety of methods can be used to remove contaminating material in order to purify the enzyme and increase its specific activity. Enzymes that are used as diagnostic reagents and in clinical therapeutics are normally prepared to a high degree of purity, because great emphasis is placed on the specificity of the reaction that is being catalysed. Clearly the higher the level of purification, the greater the cost of enzyme production. In the case of many bulk industrial enzymes the degree of purification is less important, and such enzymes may often be sold as very crude preparations of culture broth containing the growth medium, organisms (whole or fragmented) and enzymes of interest. However, even when the cheapest bulk enzymes are utilized (e.g. proteases for use in washing powders), the enzyme cost can contribute around 5–10% of the final product value.

Pretreatment

At the end of a fermentation in which a microorganism rich in the required enzyme has been cultured, the broth may be cooled rapidly to 5°C to prevent further microbial growth and stabilize the enzyme product. The pH may also be adjusted to optimize enzyme stability. If the enzyme-producing organism is a fungus, this may be removed by centrifugation at low speed. If the enzyme source is bacterial, the bacteria are often flocculated with aluminum sulfate or calcium chloride, which negate the charge on the bacterial membranes, causing them to clump and thus come out of suspension.

Extracellular enzymes are found in the liquid component of the pretreatment process. However, intracellular enzymes require more extensive treatment. The biomass may be concentrated by centrifugation and washed to remove medium components. The cellular component must then be ruptured to release the enzyme content. This can be done using one or more of the following processes:

• • ball milling (using glass beads)
• • enzymic removal of the cell wall
• • freeze–thaw cycles
• • liquid shearing through a small orifice at high pressure (e.g. within a French press)
• • osmotic shock
• • sonication.

Separation of enzymes from the resulting solution may then involve a variety of separation processes, which are often employed in a sequential fashion.

The first step in an enzyme purification procedure commonly involves separation of the proteins from the non-protein components by a process of salting out. Proteins remain in aqueous solution because of interactions between the hydrophilic (water-loving) amino acids and the surrounding water molecules (the solvent). If the ionic strength of the solvent is increased by adding an agent such as ammonium sulfate, some of the water molecules will interact with the salt ions, thereby decreasing the number of water molecules available to interact with the protein. Under such conditions, when protein molecules cannot interact with the solvent, they interact with each other, coagulating and coming out of solution in the form of a precipitate. This precipitate (containing the enzyme of interest and other proteins) can then be filtered or centrifuged, and separated from the supernatant.

Since different proteins vary in the extent to which they interact with water, it is possible to perform this process using a series of additions of ammonium sulfate, increasing the ionic strength in a stepwise fashion and removing the precipitate at each stage. Thus such fractional precipitation is not only capable of separating protein from non-protein components, but can also enable separation of the enzyme of interest from some of the other protein components.

Subsequently a wide variety of techniques may be used for further purification, and steps involving chromatography are standard practice.

Ion-exchange chromatography is often effective during the early stages of the purification process. The protein solution is added to a column containing an insoluble polymer (e.g. cellulose) that has been modified so that its ionic characteristics will determine the type of mobile ion (i.e. cation or anion) it attracts. Proteins whose net charge is opposite to that of the ion-exchange material will bind to it, whereas all other proteins will pass through the column. A subsequent change in pH or the introduction of a salt solution will alter the electrostatic forces, allowing the retained protein to be released into solution again.

Gel filtration can be utilized in the later stages of a purification protocol to separate molecules on the basis of molecular size. Columns containing a bed of cross-linked gel particles such as Sephadex are used. These gel particles exclude large protein molecules while allowing the entry of smaller molecules. Separation occurs because the larger protein molecules follow a path down the column between the Sephadex particles (occupying a smaller fraction of the column volume). Larger molecules therefore have a shorter elution time and are recovered first from the gel filtration column.

Affinity chromatography procedures can often enable purification protocols to be substantially simplified. Typically, with respect to enzyme purification, a column would be packed with a particulate stationary phase to which a ligand molecule such as a substrate analogue, inhibitor or cofactor of the enzyme of interest would be firmly bound. As the sample mixture is passed through the column, the enzyme interacts with, and binds, to the immobilised ligand, being retained within the column as all of the other components of the mixture pass through the column unrewarded. Subsequently a solution of the ligand is introduced to the column to release (elute) and thereby recover the bound enzyme from the column in a highly purified form.

Nowadays numerous alternative affinity chromatography procedures exist that are able to separate enzymes by binding to areas of the molecule away form their active site. Advances in molecular biology enable us to purify recombinant proteins, including enzymes, through affinity tagging. In a typical approach the gene for the enzyme of interest would be modified to code for a further short amino acid sequence at either the N- or C- terminal. For example, a range of polyhistidine tagging procedures are available to yield protein products with six or more consecutive histidine residues at their N- or C- terminal end. When a mixture containing the tagged protein of interest is subsequently passed through a column containing a nickel-nitrilotriacetic acid (Ni-NTA) agarose resin, the histidine residues on the recombinant protein bind to the nickel ions attached to the support resin, retaining the protein, whilst other protein and non-protein components pass through the column. Elution of the bound protein can then be accomplished by adding imidazole to the column, or by reducing the pH to 5-6 to displace the His-tagged protein from the nickel ions.

Such techniques are therefore capable of rapidly and highly effectively isolating an enzyme from a complex mixture in only one step, and typically provide protein purities of up to 95%. If more highly purified enzyme products are required, other supplemental options are also available, including various forms of preparative electrophoresis e.g. disc-gel electrophoresis and isoelectric focusing.

Finishing of enzymes

Enzymes are antigenic, and since problems occurred in the late 1960s when manufacturing workers exhibited severe allergic responses after breathing enzyme dusts, procedures have now been implemented to reduce dust formation. These involve supplying enzymes as liquids wherever possible, or increasing the particle size of dry powders from 10 μm to 200–500 μm by either prilling (mixing the enzyme with polyethylene glycol and preparing small spheres by atomization) or marumerizing (mixing the enzyme with a binder and water, extruding long filaments, converting them into spheres in a marumerizer, drying them and covering them with a waxy coat).

Industrial enzymology

Although many industrial processes, such as cheese manufacturing, have traditionally used impure enzyme sources, often from animals or plants, the development of much of modern industrial enzymology has gone hand in hand with the commercial exploitation of microbial enzymes. These were introduced to the West in around 1890 when the Japanese scientist Jokichi Takamine settled in the U.S.A. and set up an enzyme factory based on Japanese technology. The principal product was Takadiastase, a mixture of amylolytic and proteolytic enzymes prepared by cultivating the fungus Aspergillus oryzae on rice or wheat bran. Takadiastase was marketed successfully in the U.S.A. as a digestive aid for the treatment of dyspepsia, which was then believed to result from the incomplete digestion of starch.

Bacterial enzymes were developed in France by August Boidin and Jean Effront, who in 1913 found that Bacillus subtilis produced a heat-stable α-amylase when grown in a liquid medium made by extraction of malt or grain. The enzyme was primarily used within the textile industry for the removal of the starch that protects the warp in the manufacture of cotton.

In around 1930 it was found that fungal pectinases could be used in the preparation of fruit products. In subsequent years, several other hydrolases were developed and sold commercially (e.g. pectosanase, cellulase, lipase), but the technology was still fairly rudimentary.

After World War Two the fermentation industry underwent rapid development as methods for the production of antibiotics were developed. These methods were soon adapted for the production of enzymes. In the 1960s, glucoamylase was introduced as a means of hydrolysing starch, replacing acid hydrolysis. Subsequently, in the 1960s and 1970s, proteases were incorporated into detergents and then glucose isomerase was introduced to produce sweetening agents in the form of high-fructose syrups. Since the 1990s, lipases have been incorporated into washing powders, and a variety of immobilized enzyme processes have been developed (see section on enzyme immobilization), many of which utilize intracellular enzymes.

Currently, enzymes are used in four distinct fields of commerce and technology ( Table 6 ):

• • as industrial catalysts
• • as therapeutic agents
• • as analytic reagents
• • as manipulative tools (e.g. in genetics).

Of the thousands of different types of enzymes, about 95% are available from suppliers in quantities ranging from μg to kg, provided essentially for research purposes. Around 40–50 enzymes are produced on an industrial scale (i.e. ranging from multiple kilograms to tonnes per annum). The global enzyme market is currently dominated by the hydrolases, especially the proteases, together with amylases, cellulases and lipases supplied either as liquid concentrates or as powders or granules that release the soluble enzyme on dissolution. Global production is dominated by two companies, which between them supply more than two-thirds of the global enzyme market, namely the Danish company Novozymes, with a market share of 47%, and the U.S. company DuPont (which recently acquired Genencor), with 21%.

The value of the world enzyme market has increased steadily from £110 million in 1960 to £200 million in 1970, £270 million in 1980, £1 000 million in 1990 and over £2 000 million in 2010. Food and beverage enzymes represented the largest sector of the industrial enzymes market in 2010, with a value of £750 million, and the market for enzymes for technical applications (including diagnostic applications, research and biotechnology) accounted for a further £700 million. Estimates of future demand are in the range of £4 000–5 000 million between 2015 and 2016, growing at a rate of 6–7% annually. The developing economies of the Asia-Pacific Region, the Middle East and Africa are now seen to be emerging as the fastest growing markets for industrial enzymes.

Microbial enzymes are typically produced in batches by culturing the producing organism within a batch fermenter. Fermentation typically lasts between 30 and 150 h, with the optimum enzyme yield for the process falling somewhere between the optimum biomass yield and the point of maximal enzyme activity within the cells. Relatively small fermenters with a volume of 10–100 m 3 are generally employed, allowing flexibility where a number of different products are being produced. Many production systems are optimized by means of a fed-batch process, in which substrates are gradually fed into the reactor over the course of the fermentation, rather than being provided all at once at the start of the process. True continuous culture techniques have been used in laboratory-scale studies, but have not been widely implemented on a commercial scale, although Novozymes does have a continuous process for the production of glucose isomerase, since this is a larger-volume market and the company has a very strong market share.

Enzyme immobilization

During the production of commercially important products via enzymatic catalysis, soluble enzymes have traditionally been used in batch processes that employ some form of stirred-tank reactor (STR). In these processes, at the end of the batch run the product must be separated from any unused substrate, and also from the enzyme catalyst. Removal of the enzyme at this stage can be achieved by thermal denaturation (only if the product is thermostable) or by ammonium sulfate precipitation or ultrafiltration. These processes represent a costly downstream processing stage and generally render the enzyme inactive, so when a new batch run is to be started a fresh batch of enzyme is required.

Immobilized enzyme systems, in contrast, ‘fix’ the enzyme so that it can be reused many times, which has a significant impact on production costs. As a very simple example, if an enzyme is mixed with a solution of warm (but not too hot) agar and this is allowed to set, the enzyme will be entrapped (for the purposes of this example let us ignore the fact that the enzyme will gradually leak out of this gel). The agar can then be cut up into cubes and these can be placed in a STR, together with substrate, as shown in Figure 12 . Again the reaction would be allowed to proceed (and it might actually be slower due to diffusional constraints and other effects described later). At the end of the batch run the catalyst can now be easily separated from the product by passing the reactor contents through a coarse mesh. Immediately an important downstream processing step has been carried out and, just as importantly, the active enzyme has been recovered so that it can be reused for the next batch run. This ease of separation of enzyme from product is a major advantage of all immobilized systems over their counterparts that use free (i.e. soluble) enzyme.

This physical advantage of ease of reuse of immobilized biocatalysts is one of the main reasons why such systems are favoured commercially. However, immobilization may also produce biochemical changes that lead to enhanced biocatalyst stability, which may be manifested as:

• • an increased rate of catalysis
• • prolonged duration of catalysis
• • greater operational stability to extremes of pH, temperature, etc.

The particular advantage(s) conferred by immobilization will therefore differ from one system to another. It should be noted that often there may be no biochemical advantage at all, and the simple physical advantage of ease of separation of the biocatalyst from the product may be sufficient to favour the commercial development of an immobilized process.

At this point one problem that will immediately spring to mind for most students is that they have always been taught to fully mix all of the reagents of a reaction, yet the basic principle of immobilization is to partition the biocatalyst into a distinct phase, rather than mix it homogeneously with the substrate. Will this not cause reaction rates to be low? The answer to this question is yes, and the relationship between the activity of an immobilized system and a non-immobilized system can be expressed as the effectiveness factor (η), where:

Thus an immobilized system with an effectiveness factor of 0.1 would show only 10% of the activity of a non-immobilized system with the same amount of enzyme and operating under the same conditions. At first sight this might appear to be a major problem. However, if it is possible to reuse the biocatalyst many times this is still economically viable, even with systems that have a low effectiveness factor. In principle, therefore, for economic viability:

Thus if an immobilized system has an effectiveness factor of 0.1 (i.e. 10%) and we can reuse the biocatalyst 10 times, we essentially achieve the same overall catalytic activity with both the non-immobilized system and the immobilized one. However, if we are able to reuse the biocatalyst 100 times we in fact obtain 10 times more total activity from the immobilized system than from the equivalent non-immobilized system, so the immobilized system may be economically preferable.

Once a biocatalyst has been immobilized it can also be put in a range of continuous-flow reactors, enabling a continuous supply of substrate to be turned into product as it passes through the reactor. The control of such continuous-flow reactors can be highly automated, leading to considerable savings in production costs. For example, a STR can be easily modified to produce a continuous-flow stirred-tank reactor (CSTR) ( Figure 13 a), in which the enzyme is held within the reactor by a coarse mesh, and the product continuously flows out of the reactor as substrate is pumped in. It is also possible to produce a packed-bed reactor (PBR) ( Figure 13 b), in which the agar cubes are packed into a column and the substrate is pumped through the bed without any need for stirring.

CSTRs and PBRs enable the enzyme to be reused many times before it needs to be replaced. For example, in the production of high-fructose syrups, the immobilized glucose isomerase enzyme would typically be used continuously for between 2 and 4 months, and only after this time (when its activity would have dropped to 25% of the original level) would it need to be replaced.

The overall operating costs of continuous-flow reactors are often significantly lower than those of equivalent batch processes. Batch reactors need to be emptied and refilled frequently at regular intervals. Not only is this procedure expensive, but it also means that there are considerable periods of time when such reactors are not productive (so-called ‘downtime’). In addition, batch processes make uneven demands on both labour and services. They may also result in pronounced batch-to-batch variations, as the reaction conditions change with time, and they may be difficult to scale up, due to the changing power requirements for efficient mixing. Due to their higher overall process efficiency, continuous processes using immobilized enzymes may be undertaken in production facilities that are around 10 to 100 times smaller than those required for equivalent batch processes using soluble enzymes. Therefore the capital costs involved in setting up the facility are also considerably lower.

Immobilization techniques

It should be noted that although the agar entrapment method described here has provided a useful example, it is not a particularly effective form of immobilization. The high temperature required to prevent the agar from setting may lead to thermal inactivation of the enzyme, and the agar gel itself is very porous and will allow the enzyme to leak out into the surrounding solution.

There are in fact thousands of different techniques of immobilization, all of which are much more effective than our example. In general these techniques can be classified as belonging to one of three categories ( Figure 14 ):

• • adsorption
• • covalent bonding
• • entrapment.

The physical adsorption of an enzyme to a supporting matrix is the oldest method of immobilization. As early as 1916, J.M. Nelson and Edward G. Griffin described the adsorption of yeast invertase on to activated charcoal, and the subsequent use of this preparation for sucrose hydrolysis. Over the years a variety of adsorbents have been used, including cellulose, Sephadex, polystyrene, kaolinite, collagen, alumina, silica gel and glass. Such immobilization procedures are extremely easy to perform, as the adsorbent and enzyme are simply stirred together for a time (typically minutes to hours). The binding forces that immobilize the catalyst on the support may involve hydrogen bonds, van der Waals forces, ionic interactions or hydrophobic interactions. Such forces are generally weak in comparison with covalent bonds—for example, a hydrogen bond has an energy content of about 20 kJ mol −1 , compared with 200–500 kJ mol −1 for a covalent bond. Thus, when using such methods, yields (i.e. the amount of enzyme bound per unit of adsorbent) are generally low. In addition, adsorption is generally easily reversed, and can lead to desorption of the enzyme at a critical time.

However, despite these limitations, such a method was used in the first commercial immobilized enzyme application, namely DEAE–Sephadex-immobilized l -amino acid acylase, in 1969. DEAE–Sephadex is an ion-exchange resin that consists of an inert dextran particle activated by the addition of numerous diethylaminoethyl groups. Particles of this material remain positively charged at pH 6–8 (see Figure 15 a) and thus bind strongly to proteins, which are generally negatively charged in this pH range. If the pH is kept constant, the enzyme and support will remain ionically linked. However, when over time the enzyme loses its activity through denaturation, the pH can be adjusted to a more acidic value, the old enzyme will be desorbed, and the pH can then be readjusted back to pH 6–8 and a fresh batch of enzyme bound. Thus the support matrix may be used many times, giving the process significant economic benefits.

Clearly DEAE–Sephadex immobilization is only of value for enzymes that have a neutral-to-alkaline pH optimum. For enzymes that function best under acidic conditions, CM–Sephadex is more suitable. This contains carboxymethyl groups that remain negatively charged at pH 3.5–4.5 ( Figure 15 b). Proteins at this pH are generally positively charged and will thus ionically bind to the support. Desorption of the enzyme will occur when the pH is adjusted to a more alkaline value.

Due to the simplicity and controllability of this immobilization procedure, combined with the economic benefits of reuse of the support, ion-exchange materials are now widely used as the method of choice in many industrial settings.

Covalent bonding

Immobilization of enzymes by covalent bonding to activated polymers is a widely used approach since, although it is often a tedious procedure, it is capable of producing an immobilized enzyme that is firmly bound to its support. The range of polymers and chemical coupling procedures that are used is enormous.

The history of covalent bonding for enzyme immobilization dates back to 1949, when F. Michael and J. Ewers used the azide derivative of carboxymethylcellulose to immobilize a variety of proteins. Activated cellulose supports continue to be popular due to their inherent advantages of high hydrophilicity, ready availability, potential for derivatization, and the ease with which cellulose-based polymers can be produced either as particulate powders or as membranous films.

It is often more effective not to build the reactive group into the cellulose itself, but instead to use a chemical ‘bridge’ between the cellulose and the enzyme molecule. The requirements for such a bridging or linking molecule are that it must be small, and that once it has reacted with the support it must have a further reactive group capable of reacting with the enzyme. An example of such a bridging molecule is glutaraldehyde, which contains two aldehyde groups, one at either end of its (CH 2 ) 3 moiety. At neutral pH values the aldehyde groups will react with free amino groups. Thus one end of the glutaraldehyde molecule may be attached to the support, and the other to the enzyme.

Covalently immobilized enzymes are strongly bound to their support, so when the proteins denature they are difficult to remove (in contrast to adsorption, as described earlier). Therefore it is usual for both the enzyme and the support to be replaced. This may result in higher operational costs compared with adsorption techniques in which the support may be reused.

The entrapment of an enzyme can be achieved in a number of ways:

• • inclusion within the matrix of a highly cross-linked polymer
• • separation from the bulk phase by a semi-permeable ‘microcapsule’
• • dissolution in a distinct non-aqueous phase.

An important feature of entrapment techniques is that the enzyme is not in fact attached to anything. Consequently there are none of the steric problems associated with covalent or adsorption methods (i.e. the possibility of the enzyme binding in such a way that its active site is obstructed by part of the supporting polymer matrix).

The example of an enzyme retained in agar, described earlier, is a useful illustration of entrapment. A preferable alternative involves mixing the catalyst with sodium alginate gel and extruding this into a solution of calcium chloride to produce solid calcium alginate particles. This technique has the advantage of not requiring the use of high temperatures. However, although it is a popular activity in teaching laboratories, outside that setting it is generally unsuitable for the immobilization of purified enzymes, as these are often able to leak out of the gel. Entrapment techniques for purified enzymes are more likely to involve retaining the enzyme behind some form of ultrafiltration membrane. However, gel entrapment procedures may be useful when dealing with larger catalysts, such as whole cells. For example, gel-immobilized living yeast cells have been used successfully in the manufacture of champagne by Moët & Chandon.

Immobilization: changes in enzyme properties

Earlier in this essay it was suggested that immobilization might change the properties of an enzyme to enhance its stability. Initially it was believed that such enhanced stability resulted from the formation of bonds between the enzyme and the supporting matrix that physically stabilize the structure of the protein. Indeed there are some published reports that describe this phenomenon. With regard to the stabilization of proteolytic enzymes, which often exhibit more prolonged activity in the immobilized state, this is most probably explained by the fact that such proteases in free solution are prone to autodigestion (i.e. enzyme molecules cleave the peptide bonds of adjacent enzyme molecules), a process that is largely prevented when they are fixed to a supporting matrix.

However, the effects of immobilization are more often due to the supporting matrix changing the microenvironment around the enzyme and/or introducing diffusional constraints that modify the activity of the catalyst. Consider, for example, immobilization of the enzyme by adsorption on to a polyanionic (negatively charged) support such as cellulose. If the substrate is a cation (i.e. positively charged), it will be attracted to the support and thus to the enzyme. In this case the enzyme might well display higher activity, as the substrate concentration in its microenvironment would be higher than that in the surrounding bulk phase. Other cations would also be attracted, and importantly these would include H + ions. Thus the microenvironment would also be enriched in H + ions, so the pH surrounding the enzyme would be lower than the pH of the bulk phase. Consequently the enzyme would also exhibit an altered pH profile compared with that of its soluble counterpart.

In addition, the immobilization matrix might act as a barrier to the diffusion of substrates, products and other molecules. For example, if a high enzyme loading was put into a gel particle and this was then immersed in substrate solution, the substrate would diffuse into the gel and rapidly be converted into product. Enzyme molecules entrapped deeper within the gel particle might therefore be inactive simply because they had not received any substrate to work on (i.e. all of the substrate was converted to product in the outer layers of the particle). Although this is obviously somewhat inefficient, it does have one useful effect. When over time the enzyme within the system denatures, the loss of activity of the enzyme in the outer part of the particle means that substrate will now diffuse deeper into the particle to reach the previously unused core enzyme molecules. In effect this inner reserve of enzyme will offset the loss of enzyme activity through denaturation, so the system will show little or no overall loss of activity. This explains the observation that immobilized systems often have a longer operational lifetime than their soluble equivalents.

In addition, it is of interest that enzymes bound to natural cell membranes (phospholipid bilayers) within living cells will also probably demonstrate these effects, and immobilized systems thus provide useful models for the study of such membrane-bound proteins in living cells.

Immobilized enzymes at work

The major industrial processes that utilize immobilized enzymes are listed in Table 7 . Sales of immobilized enzymes peaked in 1990, when they accounted for about 20% of all industrial enzyme sales, almost entirely due to the use of glucose isomerase for the production of sweetening agents. Other commercial applications utilize penicillin acylase, fumarase, β–galactosidase and amino acid acylase. Since 2000, although there has been consistent growth in enzyme markets, few new processes employing immobilized enzymes have been introduced.

The following three examples highlight many of the biochemical, technological and economic considerations relating to the use of immobilized enzymes on a commercial and industrial scale.

Production of high-fructose syrup

Undoubtedly the most significant large-scale application of immobilized enzymes involves the production of high-fructose corn syrup (HFCS). Although most of the general public believe that sucrose is responsible for the ‘sweetness’ of food and drinks, there have been significant efforts to replace sucrose with alternative, and often cheaper, soluble caloric sweetening agents. HFCS is a soluble sweetener that has been used in many carbonated soft drinks since the 1980s, including brand-name colas such as Coca-Cola and Pepsi-Cola. HFCS is produced by the enzymatic digestion of starch derived from corn (maize). Developments in HFCS production have been most prominent in countries such as the U.S.A., which have a high capacity to produce starch in the form of corn, but which do not cultivate significant amounts of sugar cane or sugar beet, and must therefore import either the raw products (for processing) or the refined sugar (sucrose) itself.

Simple corn syrups can be manufactured by breaking down starch derived from corn using the enzyme glucoamylase alone or in combination with α-amylase. These enzymes are cheap and can be used in a soluble form. Since starch has to be extracted from corn at high temperatures (because starch has poor solubility at low temperatures and forms very viscous solutions), the process utilizes enzymes from thermophilic organisms, which have very high temperature optima. Simple corn syrup is therefore composed predominantly of glucose, which unfortunately has only 75% of the sweetness of sucrose. However, in order to make the syrup sweeter the enzyme glucose isomerase, which catalyses the following reaction, can be employed:

This enzyme (described previously in the section on properties and mechanisms of enzyme action) will produce a roughly 50:50 mixture of glucose and fructose at equilibrium, and since fructose has 150% of the sweetness of sucrose, this glucose:fructose mixture will have a similar level of sweetness to sucrose. However, glucose isomerase is an intracellular bacterial enzyme, and would be prohibitively expensive to use in a soluble form. This makes it an ideal candidate for use in an immobilized process.

The first glucose isomerase enzyme to be isolated was obtained from species of Pseudomona s in 1957, and more useful enzymes were isolated throughout the 1960s from species of Bacillus and Streptomyces. In 1967, the Clinton Corn Processing Company of Iowa, U.S.A. (later renamed CPC International) introduced a batch process that utilized an immobilized glucose isomerase enzyme, and by 1972 the company had developed a continuous process for the manufacture of HFCS containing 42% fructose using a glucose isomerase enzyme immobilized on a DEAE ion-exchange support.

During the late 1970s, advances in enzymology, process engineering and fractionation technology led to the production of syrups with a higher fructose content, and today HFCS containing 55% fructose is generally produced, and is commonly used in soft drinks, although 42% fructose syrups are still also produced for use in some processes, including the production of bakery foodstuffs.

In 2010, the U.S. production of HFCS was approximately 8 million metric tons, accounting for 37% of the U.S. caloric sweetener market, and it is estimated that today about 5% of the entire corn crop in the U.S.A. is used to produce HFCS.

Hydrolysis of lactose

Within the dairy industry the production of 1 kg of cheese requires about 10 litres of milk, and produces about 9 litres of whey as a waste product. Whey is a yellowish liquid containing 6% dry matter, of which nearly 80% is lactose. The enzyme lactase (β-galactosidase) may be used to break down lactose to its constituent monosaccharides, namely glucose and galactose, which are more soluble than lactose, and have potential uses as carbon sources in microbial fermentation, and can also be used as caloric sweeteners.

Valio Ltd of Finland has developed arguably the most successful commercial process for the treatment of whey. Using a lactase enzyme obtained from Aspergillus , immobilized by adsorption and cross-linked on to a support resin, whey syrups are produced that have been utilized as an ingredient in drinks, ice cream and confectionery products. The Aspergillus enzyme has an acid pH optimum of 3–5, and by operating at low pH the process avoids excessive microbial contamination. Treatment plants that utilize 600-litre columns have been built in Finland, and these are used to treat 80 000 litres of whey per day. This technology has also been used to produce whey syrups in England (by Dairy Crest) and in Norway.

Similar technology can also be used to remove lactose from milk. Lactose-free milk is produced for consumption by those who have lactose intolerance (a genetic condition), and also for consumption by pets such as cats, which are often unable to digest lactose easily. The first industrial processing facility to use immobilized lactase to treat milk was opened in 1975, when Centrale del Latte of Milan, Italy, utilized a batch process in which yeast ( Saccharomyces ) lactase, with a neutral pH optimum of 6–8, was immobilized within hollow permeable fibres. This process was capable of treating 10 000 litres of milk per day, and was operated at low temperature to prevent microbial contamination.

Production of semi-synthetic penicillins

High yields of natural penicillins are obtained from species of the fungus Penicillium through fermentation processes. However, over the years many microbial pathogens have become resistant to natural penicillins, and are now only treatable with semi-synthetic derivatives. These are produced through cleavage of natural penicillin, such that the G or V side chain is removed from the 6-aminopenicillanic acid (6-APA) nucleus of the molecule:

Thereafter, by attachment of a chemically different side chain, a semi-synthetic penicillin product (e.g. ampicillin, amoxicillin) can be formed. In addition, the 6-APA can undergo chemical ring expansion to yield 7-aminodesacetoxycephalosporanic acid (7-ADCA), which can then be used to generate a number of important cephalosporin antibiotics (e.g. cephalexin, cephradine, cefadroxil).

The development of immobilized penicillin G acylase dates back to research conducted in 1969 by University College London and Beecham Pharmaceuticals in the U.K. Penicillin G acylases are intracellular enzymes found in E. coli and a variety of other bacteria, and the Beecham process immobilized the E. coli enzyme on a DEAE ion-exchange support. Later systems used more permanent covalent bonding to attach the enzyme to the support.

In the 1980s and 1990s, world production of penicillins was dominated by European manufacturers, which accounted for production of around 30 000 tonnes of penicillin per annum, 75% of which was used for the manufacture of semi-synthetic penicillins and cephalosporins. However, over the past 10 years, due to increasing costs of labour, energy and raw materials, more bulk manufacturing has moved to the Far East, where China, Korea and India have become major producers. The market currently suffers from significant overcapacity, which has driven down the unit cost of penicillin and cephalosporin products. However, penicillins and cephalosporins still represent one of the world's major biotechnology markets, with annual sales of about £10 000 million, accounting for 65% of the entire global antibiotics market.

Enzymes in analysis

Enzymes have a wide variety of uses in analytical procedures. Their specificity and potency allow both detection and amplification of a target analyte. ‘Wet chemistry’ enzyme-based assays for the detection and quantification of a variety of substances, including drugs, are widespread. Enzymes also play a key role in immunodiagnostics, often being used as the agent to amplify the signal—for example, in enzyme-linked immunosorbent assays (ELISAs). Within DNA-fingerprinting technology, the enzyme DNA polymerase plays a key role in the amplification of DNA molecules in the polymerase chain reaction. However, ‘wet chemistry’ analytical methods are increasingly being replaced by the use of biosensors—that is, self-contained integrated devices which incorporate a biological recognition component (usually an immobilized enzyme) and an electrochemical detector (known as a transducer).

Much of the technological development of biosensors has been motivated by the need to measure blood glucose levels. In 2000, the World Health Organization estimated that over 170 million people had diabetes, and predicted that this figure will rise to over 360 million by 2030. In view of this, many companies have made significant investments in R&D programmes that have led to the availability of a wide variety of glucose biosensor devices.

In 1962, Leland Clark Jr coined the term ‘enzyme electrode’ to describe a device in which a traditional electrode could be modified to respond to other materials by the inclusion of a nearby enzyme layer. Clark's ideas became a commercial reality in 1975 with the successful launch of the Yellow Springs Instruments (YSI) model 23A glucose analyser. This device incorporated glucose oxidase together with a peroxide-sensitive electrode to measure the hydrogen peroxide (H 2 O 2 ) produced during the following reaction:

In this device, the rate of H 2 O 2 formation is a measure of the rate of the reaction, which depends on the concentration of glucose in solution, thus allowing the latter to be estimated.

As was discussed earlier, in enzyme-catalysed reactions the relationship between substrate concentration and reaction rate is not linear, but hyperbolic (as described by the Michaelis–Menten equation). This is also true for the glucose oxidase within a biosensor. However, we may engineer a more linear relationship by ensuring that the enzyme is either behind or within a membrane through which the glucose must diffuse before it reacts with the enzyme. This means that the system becomes diffusionally, rather than kinetically, limited, and the response is then more linearly related to the concentration of glucose in solution.

Over the years the YSI model 23A glucose analyser has been replaced by a range of much more advanced models. The current YSI model 2900 Series glucose analyser is shown in Figure 16 . This instrument has a 96-sample rack that enables batches of samples to be run, with the analysis of each sample taking less than a minute. The instrument can measure the glucose content of whole blood, plasma or serum, and requires only 10 μl of sample per analysis. The membrane-bound glucose oxidase typically only needs to be replaced every 3 weeks, thereby reducing the cost of analysis. These systems also offer advanced data-handling and data-storage facilities.

In addition, these instruments can be modified to analyse a wide variety of other substances of biological interest, simply by incorporating other oxidase enzymes into the membrane ( Table 8 ).

To enable diabetic patients to take their own blood glucose measurements, small hand-held biosensors have also been developed, which are in fact technologically more advanced because the enzyme and transducer are more intimately linked on the sensor surface. The first device of this type was launched in 1986 by Medisense, and was based on technology developed in the U.K. at Cranfield and Oxford Universities. The ExacTech blood glucose meter was the size and shape of a pen, and used disposable electrode strips. This device was followed by a credit card-style meter in 1989. Such devices again rely on glucose oxidase as the biological component, but do not measure the reaction rate via the production (and detection) of H 2 O 2 . Instead they rely on direct measurement of the rate of electron flow from glucose to the electrode surface. The reactions that occur within this device may be summarized as follows:

and at the electrode surface:

where GO x -FAD represents the FAD redox centre of glucose oxidase in its oxidized form, and GO x -FADH 2 represents the reduced form.

Basically electrons are removed from the glucose molecules and passed via the enzyme to the ferrocene mediator, which then donates them to the working electrode surface, resulting in the generation of an electrical current that is directly proportional to the rate of oxidation of glucose, and thus proportional to the glucose concentration in the sample.

Medisense, whose only product was its blood glucose meter, was bought by Abbott Diagnostics in 1996, and Abbott-branded devices continued to use and develop this technology for some time.

In 1999, Therasense marketed a glucose meter that represented the next generation of sensing technology, and integrated the enzyme even more closely with the electrode. Originally developed by Adam Heller at the University of Texas in the 1990s, wired-enzyme electrodes do not rely on a soluble mediator such as the ferrocene used in the Medisense devices. Instead the enzyme is immobilized in an osmium-based polyvinyl imidazole hydrogel in which the electrons are passed from enzyme to electrode by a series of fixed electroactive osmium centres that shuttle the electrons onward in a process called ‘electron hopping.’

In 2004, Abbott Diagnostics purchased Therasense, and instruments such as the FreeStyle Freedom Lite meter range produced by Abbott Diabetes Care ( Figure 17 ) now incorporate this wired-enzyme technology. Devices of this type are highly amenable to miniaturization.

Continuous measuring devices are becoming increasingly available, and may well revolutionize the control of certain disease conditions. For example, with regard to diabetes, devices such as the FreeStyle Navigator range from Abbott Diabetes Care use the same wired-enzyme technology as that described earlier, but now incorporate this into a tiny filament about the diameter of a thin hypodermic needle. This is inserted approximately 5 mm under the skin to measure the glucose level in the interstitial fluid that flows between the cells. The unit is designed to remain in situ for up to 5 days, during which time it can measure the glucose concentration every minute. A wireless transmitter sends the glucose readings to a separate receiver anywhere within a 30-metre range, and this can then issue an early warning alarm to alert the user to a falling or rising glucose level in time for them to take appropriate action and avoid a hypoglycaemic or hyperglycaemic episode.

In addition, experimental units have already been developed that link continuous glucose biosensor measurement systems with pumps capable of gradually dispensing insulin such that the diabetic condition is automatically and reliably controlled, thereby avoiding the traditional peaks and troughs in glucose levels that occur with conventional glucose measurement and the intermittent administration of insulin.

Therefore, looking to the future, we may confidently expect to see the development of biosensor systems that can continuously monitor a range of physiologically important analytes and automatically dispense the required medication to alleviate the symptoms of a number of long-term chronic human illnesses.

Closing remarks

For the sake of conciseness, this guide has been limited to some of the basic principles of enzymology, together with an overview of the biotechnological applications of enzymes. It is important to understand the relationship between proteins and the nucleic acids (DNA and RNA) that provide the blueprint for the assembly of proteins within the cell. Genetic engineering is thus predominantly concerned with modifying the proteins that a cell contains, and genetic defects (in medicine) generally relate to the abnormalities that occur in the proteins within cells. Much of the molecular age of biochemistry is therefore very much focused on the study of the cell, its enzymes and other proteins, and their functions.

Abbreviations

This article is a reviewed, revised and updated version of the following ‘Biochemistry Across the School Curriculum’ (BASC) booklet: Teal A.R. & Wymer P.E.O., 1995: Enzymes and their Role in Technology. For further information and to provide feedback on this or any other Biochemical Society education resource, please contact gro.yrtsimehcoib@noitacude . For further information on other Biochemical Society publications, please visit www.biochemistry.org/publications .

Recommended reading and key publications

1. historically important landmark papers (in chronological order).

• Takamine J. Process of making diastatic enzyme. 1894 U.S. Pat. 525,823. Describes the first commercial exploitation of semi-purified enzymes in the West. [ Google Scholar ]
• Briggs G.E., Haldane J.B.S. A note on the kinetics of enzyme action. Biochem. J. 1925; 19 :338–339. A classic paper in which the steady-state assumption was introduced into the derivation of the Michaelis–Menten equation. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Koshland D.E., Jr Application of a theory of enzyme specificity to protein synthesis. Proc. Natl Acad. Sci. U.S.A. 1958; 44 :98–104. Describes the proposal of an ‘induced fit’ mechanism of substrate binding. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Clark L.C., Jr, Lyons C. Electrode systems for continuous monitoring in cardiovascular surgery. Ann. N.Y. Acad. Sci. 1962; 102 :29–45. Introduces the concept of a biosensor for measuring blood glucose levels during surgery. [ PubMed ] [ Google Scholar ]
• Monod J., Wyman H., Changeux J.P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 1965; 12 :88–118. Describes the ‘concerted’ model of transitions of allosteric proteins in which all constituent monomers are in either the T-state or the R-state. [ PubMed ] [ Google Scholar ]
• Koshland D.E., Jr, Némethy G., Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. 1966; 5 :365–385. Describes the ‘sequential’ model of transitions of allosteric proteins in which protein moves through hybrid structures with some monomers in the T-state and some in the R-state. [ PubMed ] [ Google Scholar ]
• Updike S.J., Hicks G.P. The enzyme electrode. Nature. 1967; 214 :986–988. Describes the simplification of the electrochemical assay of glucose by immobilizing and thereby stabilizing the glucose oxidase enzyme. [ PubMed ] [ Google Scholar ]
• Tramontano A., Janda K.D., Lerner R.A. Catalytic antibodies. Science. 1986; 234 :1566–1570. [ PubMed ] [ Google Scholar ]
• Pollack S.J., Jacobs J.W., Schultz P.G. Selective chemical catalysis by an antibody. Science. 1986; 234 :1570–1573. The first reports of antibody proteins that demonstrate catalytic activity. [ PubMed ] [ Google Scholar ]
• Johnson K.A., Goody R.S. The original Michaelis constant: translation of the 1913 Michaelis–Menten paper. Biochemistry. 2011; 50 :8264–8269. A modern translation, commentary and re-analysis of the original 1913 paper, Die Kinetik der Invertinwirkung. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Taylor A.I., Pinheiro V.B., Smola M.J., Morgunov A.S., Peak-Chew S., Cozens C., Weeks K.M., Herdewijn P., Holliger P. Catalysts from synthetic genetic polymers. Nature. 2015; 518 :427–430. Describes the first artificial enzymes to be created using synthetic biology. [ PMC free article ] [ PubMed ] [ Google Scholar ]

2. Enzyme principles

• Changeux J.-P. 50 years of allosteric interactions: the twists and turns of the models. Nat. Rev. Mol. Cell Biol. 2013; 14 :819–829. [ PubMed ] [ Google Scholar ]
• Kamata K., Mitsuya M., Nishimura T., Eiki J., Nagata Y. Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase. Structure. 2004; 12 :429–438. [ PubMed ] [ Google Scholar ]

3. Enzyme applications

• Adrio J.L., Demain A.L. Microbial enzymes: tools for biotechnological processes. Biomolecules. 2014; 4 :117–139. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Clarke S.F., Foster J.R. A history of blood glucose meters and their role in self-monitoring of diabetes mellitus. Br. J. Biomed. Sci. 2012; 69 :83–93. [ PubMed ] [ Google Scholar ]
• Fernandes P. Enzymes in food processing: a condensed overview on strategies for better biocatalysts. Enzyme Res. 2010; 2010 :862537. [ PMC free article ] [ PubMed ] [ Google Scholar ]
• Vashist S.K., Zheng D., Al-Rubeaan K., Luong J.H.T., Sheu F.-S. Technology behind commercial devices for blood glucose monitoring in diabetes management: a review. Anal. Chim. Acta. 2011; 703 :124–136. [ PubMed ] [ Google Scholar ]
• Vellard M. The enzyme as drug: application of enzymes as pharmaceuticals. Curr. Opin. Biotechnol. 2003; 14 :444–450. [ PubMed ] [ Google Scholar ]
• Woodley J.M. New opportunities for biocatalysis: making pharmaceutical processes greener. Trends Biotechnol. 2008; 26 :321–327. [ PubMed ] [ Google Scholar ]

4. Useful textbooks

• Bisswanger H. Enzyme Kinetics: Principles and Methods. 2nd edn. Weinheim, Germany: Wiley-VCH; 2008. Available online and as hard copy. A user-friendly and comprehensive treatise on enzyme kinetics. [ Google Scholar ]
• Buchholz K., Kasche V., Bornscheuer U.T. Biocatalysts and Enzyme Technology. 2nd edn. Weinheim, Germany: Wiley-VCH; 2012. Best-selling textbook that provides an instructive and comprehensive overview of our current knowledge of biocatalysis and enzyme technology. [ Google Scholar ]
• Copeland R.A. In: Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists. 2nd edn. Hoboken NJ., editor. John Wiley & Sons, Inc; 2013. Provides thorough coverage of both the principles and applications of enzyme inhibitors. [ PubMed ] [ Google Scholar ]
• McGrath M.J., Scanaill C.N. Sensor Technologies: Healthcare, Wellness and Environmental Applications. New York: Apress Media, LLC; 2014. Available online. Covers sensor technologies and their clinical applications, together with broader applications that are relevant to wellness, fitness, lifestyle and the environment. [ Google Scholar ]
• Trevan M.D. Immobilized Enzymes: An Introduction and Applications in Biotechnology. Chichester: John Wiley & Sons; 1980. An older text, and difficult to find except in libraries, but it provides an introductory text for non-experts, and as yet there is no other book that fulfils this role. [ Google Scholar ]
• Whitehurst R.J., van Oort M. Enzymes in Food Technology. 2nd edn. Chichester: Wiley-Blackwell; 2009. Provides comprehensive coverage of the widespread use of enzymes in food-processing improvement and innovation. [ Google Scholar ]

Factors Affecting Enzyme Activity

Investigating rates of enzyme-controlled reactions.

Changes to the tertiary structure of an enzyme through changing the pH or temperature will affect how fast reactions are catalysed.

Temperature

• Increasing the temperature will increase the kinetic energy of the molecules.
• This increases the chance of a collision between the enzyme and substrate and so more collisions are likely in a set period of time. In other words, the rate of reaction is faster.
• Increasing the temperature by 10 o C will approximately double the rate of reaction for most enzyme-controlled reactions.

• Changing the pH changes the number of hydroxide ions and hydrogen ions (OH − and H + ) surrounding the enzyme.
• These interact with the charges on the enzyme’s amino acids, affecting hydrogen bonding and ionic bonding, so resulting in changes to the tertiary structure.

Denatured enzymes

• At extreme temperatures and pH values, the enzyme's structure may be changed. This is called a denatured enzyme.

Enzyme and Substrate Concentration

Reaction rate is influenced by the relative enzyme and substrate concentrations.

Enzyme concentration

• Increasing the concentration of enzyme in a solution means there are more enzyme molecules available to catalyse the substrate in a given amount of time

Substrate concentration

• Increasing the concentration of the substrate increases the numbers of substrate molecules that can form enzyme-substrate (ES) complexes at any one time.
• This increases the initial rate of reaction but when all the enzyme molecules are engaged in ES complexes the rate cannot increase any further.
• The rate will then plateau because the enzyme is said to be saturated.

Inhibition of Enzyme Activity

Reaction rate is influenced by the presence of competitive and non-competitive inhibitors.

Function of competitive inhibitors

• Inhibitors are chemicals that slow down the rate or stop the reaction altogether.
• Enzyme-substrate complexes cannot be formed or are formed at a much lower rate.

Structure of competitive inhibitors

• Competitive inhibitors are similar in shape to the usual substrate and affect the active site directly, blocking access for the formation of ES complexes.
• Malonate ions are similar in shape to succinate ions and act as a competitive inhibitor of succinate dehydrogenase, an important enzyme in the Krebs cycle.

Function of non-competitive inhibitors

• E.g. Lead denatures a number of enzymes required to synthesise haemoglobin.

Structure of non-competitive inhibitors

• Non-competitive inhibitors affect another part of the enzyme molecule causing a change to the shape of the active site.
• The active site is no longer complementary to the substrate molecules.

1 Biological Molecules

1.1 Monomers & Polymers

1.1.1 Monomers & Polymers

1.1.2 Condensation & Hydrolysis Reactions

1.2 Carbohydrates

1.2.1 Structure of Carbohydrates

1.2.2 Types of Polysaccharides

1.2.3 End of Topic Test - Monomers, Polymers and Carbs

1.2.4 Exam-Style Question - Carbohydrates

1.2.5 A-A* (AO3/4) - Carbohydrates

1.3.1 Triglycerides & Phospholipids

1.3.2 Types of Fatty Acids

1.3.3 Testing for Lipids

1.3.4 Exam-Style Question - Fats

1.3.5 A-A* (AO3/4) - Lipids

1.4 Proteins

1.4.1 The Peptide Chain

1.4.2 Investigating Proteins

1.4.3 Primary & Secondary Protein Structure

1.4.4 Tertiary & Quaternary Protein Structure

1.4.5 Enzymes

1.4.6 Factors Affecting Enzyme Activity

1.4.7 Enzyme-Controlled Reactions

1.4.8 End of Topic Test - Lipids & Proteins

1.4.9 A-A* (AO3/4) - Enzymes

1.4.10 A-A* (AO3/4) - Proteins

1.5 Nucleic Acids

1.5.1 DNA & RNA

1.5.2 Polynucleotides

1.5.3 DNA Replication

1.5.4 Exam-Style Question - Nucleic Acids

1.5.5 A-A* (AO3/4) - Nucleic Acids

1.6.1 Structure of ATP

1.6.2 End of Topic Test - Nucleic Acids & ATP

1.7.1 Structure & Function of Water

1.7.2 A-A* (AO3/4) - Water

1.8 Inorganic Ions

1.8.1 Inorganic Ions

1.8.2 End of Topic Test - Water & Inorganic Ions

2.1 Cell Structure

2.1.1 Introduction to Cells

2.1.2 Eukaryotic Cells & Organelles

2.1.3 Eukaryotic Cells & Organelles 2

2.1.4 Prokaryotes

2.1.5 A-A* (AO3/4) - Organelles

2.1.6 Methods of Studying Cells

2.1.7 Microscopes

2.1.8 End of Topic Test - Cell Structure

2.1.9 Exam-Style Question - Cells

2.1.10 A-A* (AO3/4) - Cells

2.2 Mitosis & Cancer

2.2.1 Mitosis

2.2.2 Investigating Mitosis

2.2.3 Cancer

2.2.4 A-A* (AO3/4) - The Cell Cycle

2.3 Transport Across Cell Membrane

2.3.1 Cell Membrane Structure

2.3.2 A-A* (AO3/4) - Membrane Structure

2.3.3 Diffusion

2.3.4 Osmosis

2.3.5 Active Transport

2.3.6 End of Topic Test - Mitosis, Cancer & Transport

2.3.7 Exam-Style Question - Membranes

2.3.8 A-A* (AO3/4) - Membranes & Transport

2.3.9 A-A*- Mitosis, Cancer & Transport

2.4 Cell Recognition & the Immune System

2.4.1 Immune System

2.4.2 The Immune Response

2.4.3 Antibodies

2.4.4 Primary & Secondary Response

2.4.5 Vaccines

2.4.7 Ethical Issues

2.4.8 End of Topic Test - Immune System

2.4.9 Exam-Style Question - Immune System

2.4.10 A-A* (AO3/4) - Immune System

3 Substance Exchange

3.1 Surface Area to Volume Ratio

3.1.1 Size & Surface Area

3.1.2 A-A* (AO3/4) - Cell Size

3.2 Gas Exchange

3.2.1 Single-Celled Organisms

3.2.2 Multicellular Organisms

3.2.3 Control of Water Loss

3.2.4 Human Gas Exchange

3.2.5 Ventilation

3.2.6 Dissection

3.2.7 Measuring Gas Exchange

3.2.8 Lung Disease

3.2.9 Lung Disease Data

3.2.10 End of Topic Test - Gas Exchange

3.2.11 A-A* (AO3/4) - Gas Exchange

3.3 Digestion & Absorption

3.3.1 Overview of Digestion

3.3.2 Digestion in Mammals

3.3.3 Absorption

3.3.4 End of Topic Test - Substance Exchange & Digestion

3.3.5 A-A* (AO3/4) - Substance Ex & Digestion

3.4 Mass Transport

3.4.1 Haemoglobin

3.4.2 Oxygen Transport

3.4.3 The Circulatory System

3.4.4 The Heart

3.4.5 Blood Vessels

3.4.6 Cardiovascular Disease

3.4.7 Heart Dissection

3.4.8 Xylem

3.4.9 Phloem

3.4.10 Investigating Plant Transport

3.4.11 End of Topic Test - Mass Transport

3.4.12 A-A* (AO3/4) - Mass Transport

4 Genetic Information & Variation

4.1 DNA, Genes & Chromosomes

4.1.2 Genes

4.1.3 A-A* (AO3/4) - DNA

4.2 DNA & Protein Synthesis

4.2.1 Protein Synthesis

4.2.2 Transcription & Translation

4.2.3 End of Topic Test - DNA, Genes & Protein Synthesis

4.2.4 Exam-Style Question - Protein Synthesis

4.2.5 A-A* (AO3/4) - Coronavirus Translation

4.2.6 A-A* (AO3/4) - Transcription

4.2.7 A-A* (AO3/4) - Translation

4.3 Mutations & Meiosis

4.3.1 Mutations

4.3.2 Meiosis

4.3.3 A-A* (AO3/4) - Meiosis

4.3.4 Meiosis vs Mitosis

4.3.5 End of Topic Test - Mutations, Meiosis

4.3.6 A-A* (AO3/4) - DNA,Genes, CellDiv & ProtSynth

4.4 Genetic Diversity & Adaptation

4.4.1 Genetic Diversity

4.4.2 Natural Selection

4.4.3 A-A* (AO3/4) - Natural Selection

4.4.4 Adaptations

4.4.5 Investigating Natural Selection

4.4.6 End of Topic Test - Genetic Diversity & Adaptation

4.4.7 A-A* (AO3/4) - Genetic Diversity & Adaptation

4.5 Species & Taxonomy

4.5.1 Classification

4.5.2 DNA Technology

4.5.3 A-A* (AO3/4) - Species & Taxonomy

4.6 Biodiversity Within a Community

4.6.1 Biodiversity

4.6.2 Agriculture

4.6.3 End of Topic Test - Species,Taxonomy& Biodiversity

4.6.4 A-A* (AO3/4) - Species,Taxon&Biodiversity

4.7 Investigating Diversity

4.7.1 Genetic Diversity

4.7.2 Quantitative Investigation

5 Energy Transfers (A2 only)

5.1 Photosynthesis

5.1.1 Overview of Photosynthesis

5.1.2 Light-Dependent Reaction

5.1.3 Light-Independent Reaction

5.1.4 A-A* (AO3/4) - Photosynthesis Reactions

5.1.5 Limiting Factors

5.1.6 Photosynthesis Experiments

5.1.7 End of Topic Test - Photosynthesis

5.1.8 A-A* (AO3/4) - Photosynthesis

5.2 Respiration

5.2.1 Overview of Respiration

5.2.2 Anaerobic Respiration

5.2.3 A-A* (AO3/4) - Anaerobic Respiration

5.2.4 Aerobic Respiration

5.2.5 Respiration Experiments

5.2.6 End of Topic Test - Respiration

5.2.7 A-A* (AO3/4) - Respiration

5.3 Energy & Ecosystems

5.3.1 Biomass

5.3.2 Production & Productivity

5.3.3 Agricultural Practices

5.4 Nutrient Cycles

5.4.1 Nitrogen Cycle

5.4.2 Phosphorous Cycle

5.4.3 Fertilisers & Eutrophication

5.4.4 End of Topic Test - Nutrient Cycles

5.4.5 A-A* (AO3/4) - Energy,Ecosystems&NutrientCycles

6 Responding to Change (A2 only)

6.1 Nervous Communication

6.1.1 Survival

6.1.2 Plant Responses

6.1.3 Animal Responses

6.1.4 Reflexes

6.1.5 End of Topic Test - Reflexes, Responses & Survival

6.1.6 Receptors

6.1.7 The Human Retina

6.1.8 Control of Heart Rate

6.1.9 End of Topic Test - Receptors, Retina & Heart Rate

6.2 Nervous Coordination

6.2.1 Neurones

6.2.2 Action Potentials

6.2.3 Speed of Transmission

6.2.4 End of Topic Test - Neurones & Action Potentials

6.2.5 Synapses

6.2.6 Types of Synapse

6.2.7 Medical Application

6.2.8 End of Topic Test - Synapses

6.2.9 A-A* (AO3/4) - Nervous Comm&Coord

6.3 Muscle Contraction

6.3.1 Skeletal Muscle

6.3.2 Sliding Filament Theory

6.3.3 Contraction

6.3.4 Slow & Fast Twitch Fibres

6.3.5 End of Topic Test - Muscles

6.3.6 A-A* (AO3/4) - Muscle Contraction

6.4 Homeostasis

6.4.1 Overview of Homeostasis

6.4.2 Blood Glucose Concentration

6.4.3 Controlling Blood Glucose Concentration

6.4.4 End of Topic Test - Blood Glucose

6.4.5 Primary & Secondary Messengers

6.4.6 Diabetes Mellitus

6.4.7 Measuring Glucose Concentration

6.4.8 Osmoregulation

6.4.9 Controlling Blood Water Potential

6.4.11 End of Topic Test - Diabetes & Osmoregulation

6.4.12 A-A* (AO3/4) - Homeostasis

7 Genetics & Ecosystems (A2 only)

7.1 Genetics

7.1.1 Key Terms in Genetics

7.1.2 Inheritance

7.1.3 Linkage

7.1.4 Multiple Alleles & Epistasis

7.1.5 Chi-Squared Test

7.1.6 End of Topic Test - Genetics

7.1.7 A-A* (AO3/4) - Genetics

7.2 Populations

7.2.1 Populations

7.2.2 Hardy-Weinberg Principle

7.3 Evolution

7.3.1 Variation

7.3.2 Natural Selection & Evolution

7.3.3 End of Topic Test - Populations & Evolution

7.3.4 Types of Selection

7.3.5 Types of Selection Summary

7.3.6 Overview of Speciation

7.3.7 Causes of Speciation

7.3.8 Diversity

7.3.9 End of Topic Test - Selection & Speciation

7.3.10 A-A* (AO3/4) - Populations & Evolution

7.4 Populations in Ecosystems

7.4.1 Overview of Ecosystems

7.4.2 Niche

7.4.3 Population Size

7.4.4 Investigating Population Size

7.4.5 End of Topic Test - Ecosystems & Population Size

7.4.6 Succession

7.4.7 Conservation

7.4.8 End of Topic Test - Succession & Conservation

7.4.9 A-A* (AO3/4) - Ecosystems

8 The Control of Gene Expression (A2 only)

8.1 Mutation

8.1.1 Mutations

8.1.2 Effects of Mutations

8.1.3 Causes of Mutations

8.2 Gene Expression

8.2.1 Stem Cells

8.2.2 Stem Cells in Disease

8.2.3 End of Topic Test - Mutation & Gene Epression

8.2.4 A-A* (AO3/4) - Mutation & Stem Cells

8.2.5 Regulating Transcription

8.2.6 Epigenetics

8.2.7 Epigenetics & Disease

8.2.8 Regulating Translation

8.2.9 Experimental Data

8.2.10 End of Topic Test - Transcription & Translation

8.2.11 Tumours

8.2.12 Correlations & Causes

8.2.13 Prevention & Treatment

8.2.14 End of Topic Test - Cancer

8.2.15 A-A* (AO3/4) - Gene Expression & Cancer

8.3 Genome Projects

8.3.1 Using Genome Projects

8.4 Gene Technology

8.4.1 Recombinant DNA

8.4.2 Producing Fragments

8.4.3 Amplification

8.4.4 End of Topic Test - Genome Project & Amplification

8.4.5 Using Recombinant DNA

8.4.6 Medical Diagnosis

8.4.7 Genetic Fingerprinting

8.4.8 End of Topic Test - Gene Technologies

8.4.9 A-A* (AO3/4) - Gene Technology

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19.5: Effect of Concentration on Enzyme Activity

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Learning Objectives

• To describe how pH, temperature, and the concentration of an enzyme and its substrate influence enzyme activity.

The single most important property of enzymes is the ability to increase the rates of reactions occurring in living organisms, a property known as catalytic activity . Because most enzymes are proteins, their activity is affected by factors that disrupt protein structure, as well as by factors that affect catalysts in general. Factors that disrupt protein structure include temperature and pH; factors that affect catalysts in general include reactant or substrate concentration and catalyst or enzyme concentration. The activity of an enzyme can be measured by monitoring either the rate at which a substrate disappears or the rate at which a product forms.

Substrate Concentration

In the presence of a given amount of enzyme, the rate of an enzymatic reaction increases as the substrate concentration increases until a limiting rate is reached, after which further increase in the substrate concentration produces no significant change in the reaction rate (part (a) of Figure \(\PageIndex{1}\)). At this point, so much substrate is present that essentially all of the enzyme active sites have substrate bound to them. In other words, the enzyme molecules are saturated with substrate. The excess substrate molecules cannot react until the substrate already bound to the enzymes has reacted and been released (or been released without reacting).

Let’s consider an analogy. Ten taxis (enzyme molecules) are waiting at a taxi stand to take people (substrate) on a 10-minute trip to a concert hall, one passenger at a time. If only 5 people are present at the stand, the rate of their arrival at the concert hall is 5 people in 10 minutes. If the number of people at the stand is increased to 10, the rate increases to 10 arrivals in 10 minutes. With 20 people at the stand, the rate would still be 10 arrivals in 10 minutes. The taxis have been “saturated.” If the taxis could carry 2 or 3 passengers each, the same principle would apply. The rate would simply be higher (20 or 30 people in 10 minutes) before it leveled off.

Enzyme Concentration

When the concentration of the enzyme is significantly lower than the concentration of the substrate (as when the number of taxis is far lower than the number of waiting passengers), the rate of an enzyme-catalyzed reaction is directly dependent on the enzyme concentration (part (b) of Figure \(\PageIndex{1}\)). This is true for any catalyst; the reaction rate increases as the concentration of the catalyst is increased.

Effect of Temperature on Activity

A general rule of thumb for most chemical reactions is that a temperature rise of 10°C approximately doubles the reaction rate. To some extent, this rule holds for all enzymatic reactions. After a certain point, however, an increase in temperature causes a decrease in the enzyme reaction rate, due to denaturation of the protein structure and disruption of the active site (part (a) of Figure \(\PageIndex{2}\)). For many proteins, denaturation occurs between 45°C and 55°C. Furthermore, even though an enzyme may appear to have a maximum reaction rate between 40°C and 50°C, most biochemical reactions are carried out at lower temperatures because enzymes are not stable at these higher temperatures and will denature after a few minutes.

At 0°C and 100°C, the rate of enzyme-catalyzed reactions is nearly zero. This fact has several practical applications. We sterilize objects by placing them in boiling water, which denatures the enzymes of any bacteria that may be in or on them. We preserve our food by refrigerating or freezing it, which slows enzyme activity. When animals go into hibernation in winter, their body temperature drops, decreasing the rates of their metabolic processes to levels that can be maintained by the amount of energy stored in the fat reserves in the animals’ tissues.

Effect of Hydrogen Ion Concentration (pH) on Activity

Because most enzymes are proteins, they are sensitive to changes in the hydrogen ion concentration or pH. Enzymes may be denatured by extreme levels of hydrogen ions (whether high or low); any change in pH, even a small one, alters the degree of ionization of an enzyme’s acidic and basic side groups and the substrate components as well. Ionizable side groups located in the active site must have a certain charge for the enzyme to bind its substrate. Neutralization of even one of these charges alters an enzyme’s catalytic activity.

An enzyme exhibits maximum activity over the narrow pH range in which a molecule exists in its properly charged form. The median value of this pH range is called the optimum pH of the enzyme (part (b) of Figure \(\PageIndex{2}\)). With the notable exception of gastric juice (the fluids secreted in the stomach), most body fluids have pH values between 6 and 8. Not surprisingly, most enzymes exhibit optimal activity in this pH range. However, a few enzymes have optimum pH values outside this range. For example, the optimum pH for pepsin, an enzyme that is active in the stomach, is 2.0.

Initially, an increase in substrate concentration leads to an increase in the rate of an enzyme-catalyzed reaction. As the enzyme molecules become saturated with substrate, this increase in reaction rate levels off. The rate of an enzyme-catalyzed reaction increases with an increase in the concentration of an enzyme. At low temperatures, an increase in temperature increases the rate of an enzyme-catalyzed reaction. At higher temperatures, the protein is denatured, and the rate of the reaction dramatically decreases. An enzyme has an optimum pH range in which it exhibits maximum activity.

Concept Review Exercises

• The concentration of substrate X is low. What happens to the rate of the enzyme-catalyzed reaction if the concentration of X is doubled?
• What effect does an increase in the enzyme concentration have on the rate of an enzyme-catalyzed reaction?
• If the concentration of the substrate is low, increasing its concentration will increase the rate of the reaction.
• An increase in the amount of enzyme will increase the rate of the reaction (provided sufficient substrate is present).

In non-enzyme-catalyzed reactions, the reaction rate increases as the concentration of reactant is increased. In an enzyme-catalyzed reaction, the reaction rate initially increases as the substrate concentration is increased but then begins to level off, so that the increase in reaction rate becomes less and less as the substrate concentration increases. Explain this difference.

Why do enzymes become inactive at very high temperatures?

An enzyme has an optimum pH of 7.4. What is most likely to happen to the activity of the enzyme if the pH drops to 6.3? Explain.

An enzyme has an optimum pH of 7.2. What is most likely to happen to the activity of the enzyme if the pH increases to 8.5? Explain.

In an enzyme-catalyzed reaction, the substrate binds to the enzyme to form an enzyme-substrate complex. If more substrate is present than enzyme, all of the enzyme binding sites will have substrate bound, and further increases in substrate concentration cannot increase the rate.

The activity will decrease; a pH of 6.3 is more acidic than 7.4, and one or more key groups in the active site may bind a hydrogen ion, changing the charge on that group.

• Biological Molecules

Factors affecting Enzyme Activity

• The activity of an Enzyme is affected by its environmental conditions . Changing these alter the rate of reaction caused by the enzyme. In nature, organisms adjust the conditions of their enzymes to produce an Optimum rate of reaction , where necessary , or they may have enzymes which are adapted to function well in extreme conditions where they live.

Temperature

Increasing temperature increases the Kinetic Energy that molecules possess. In a fluid , this means that there are more random collisions between molecules per unit time.

Since enzymes catalyse reactions by randomly colliding with Substrate molecules , increasing temperature increases the rate of reaction , forming more product.

However, increasing temperature also increases the Vibrational Energy that molecules have, specifically in this case enzyme molecules , which puts strain on the bonds that hold them together.

As temperature increases, more bonds , especially the weaker Hydrogen and Ionic bonds, will break as a result of this strain. Breaking bonds within the enzyme will cause the Active Site to change shape .

This change in shape means that the Active Site is less Complementary to the shape of the Substrate , so that it is less likely to catalyse the reaction. Eventually, the enzyme will become Denatured and will no longer function .

As temperature increases , more enzymes’ molecules’ Active Sites’ shapes will be less Complementary to the shape of their Substrate , and more enzymes will be Denatured . This will decrease the rate of reaction .

In summary, as temperature increases , initially the rate of reaction will increase , because of increased Kinetic Energy . However, the effect of bond breaking will become greater and greater , and the rate of reaction will begin to decrease .

• The temperature at which the maximum rate of reaction occurs is called the enzyme’s Optimum Temperature . This is different for different enzymes . Most enzymes in the human body have an Optimum Temperature of around 37.0 °C.

pH - Acidity and Basicity

pH measures the Acidity and Basicity of a solution. It is a measure of the Hydrogen Ion ( H + ) concentration , and therefore a good indicator of the Hydroxide Ion ( OH - ) concentration. It ranges from pH1 to pH14 . Lower pH values mean higher H + concentrations and lower OH - concentrations.

Acid solutions have pH values below 7 , and Basic solutions (alkalis are bases) have pH values above 7 . Deionised water is pH7 , which is termed ‘ neutral ’.

H + and OH - Ions are charged and therefore interfere with Hydrogen and Ionic bonds that hold together an enzyme, since they will be attracted or repelled by the charges created by the bonds. This interference causes a change in shape of the enzyme , and importantly, its Active Site .

Different enzymes have different Optimum pH values . This is the pH value at which the bonds within them are influenced by H + and OH - Ions in such a way that the shape of their Active Site is the most Complementary to the shape of their Substrate . At the Optimum pH, the rate of reaction is at an optimum.

Any change in pH above or below the Optimum will quickly cause a decrease in the rate of reaction, since more of the enzyme molecules will have Active Sites whose shapes are not (or at least are less) Complementary to the shape of their Substrate .

Small changes in pH above or below the Optimum do not cause a permanent change to the enzyme, since the bonds can be reformed . However, extreme changes in pH can cause enzymes to Denature and permanently lose their function.

Enzymes in different locations have different Optimum pH values since their environmental conditions may be different. For example, the enzyme Pepsin functions best at around pH2 and is found in the stomach, which contains Hydrochloric Acid (pH2).

Concentration

Changing the Enzyme and Substrate concentrations affect the rate of reaction of an enzyme-catalysed reaction. Controlling these factors in a cell is one way that an organism regulates its enzyme activity and so its Metabolism .

Changing the concentration of a substance only affects the rate of reaction if it is the limiting factor : that is, it the factor that is stopping a reaction from preceding at a higher rate .

If it is the limiting factor , increasing concentration will increase the rate of reaction up to a point , after which any increase will not affect the rate of reaction. This is because it will no longer be the limiting factor and another factor will be limiting the maximum rate of reaction.

As a reaction proceeds , the rate of reaction will decrease , since the Substrate will get used up . The highest rate of reaction, known as the Initial Reaction Rate is the maximum reaction rate for an enzyme in an experimental situation .

Substrate Concentration

Increasing Substrate Concentration increases the rate of reaction. This is because more substrate molecules will be colliding with enzyme molecules , so more product will be formed.

However, after a certain concentration , any increase will have no effect on the rate of reaction, since Substrate Concentration will no longer be the limiting factor . The enzymes will effectively become saturated , and will be working at their maximum possible rate .

Enzyme Concentration

Increasing Enzyme Concentration will increase the rate of reaction, as more enzymes will be colliding with substrate molecules.

However, this too will only have an effect up to a certain concentration , where the Enzyme Concentration is no longer the limiting factor .

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17.8: Enzymes

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Lab Objectives

At the conclusion of the lab, the student should be able to:

• define the following terms: metabolism, reactant, product, substrate, enzyme, denature
• describe what the active site of an enzyme is (be sure to include information regarding the relationship of the active site to the substrate)
• describe the specific action of the enzyme catalase, include the substrate and products of the reaction
• list what organelle catalase can be found in every plant or animal cell
• list the factors that can affect the rate of a chemical reaction and enzyme activity
• explain why enzymes have an optimal pH and temperature to ensure greatest activity (greatest functioning) of the enzyme (be sure to consider how virtually all enzymes are proteins and the impact that temperature and pH may have on protein function)
• explain why the same type of chemical reaction performed at different temperatures revealed different results/enzyme activity
• explain why warm temperatures (but not boiling) typically promote enzyme activity but cold temperature typically
• decreases enzyme activity
• explain why increasing enzyme concentration promotes enzyme activity
• explain why the optimal pH of a particular enzyme promotes its activity
• if given the optimal conditions for a particular enzyme, indicate which experimental conditions using that particular enzyme would show the greatest and least enzyme activity

A SlideShare element has been excluded from this version of the text. You can view it online here: pb.libretexts.org/bio1lm/?p=142

Introduction

Hydrogen peroxide is a toxic product of many chemical reactions that occur in living things. Although it is produced in small amounts, living things must detoxify this compound and break down hydrogen peroxide into water and oxygen, two non-harmful molecules. The organelle responsible for destroying hydrogen peroxide is the peroxisome using the enzyme catalase. Both plants and animals have peroxisomes with catalase. The catalase sample for today’s lab will be from a potato.

Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not consumed in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by lowering the activation energy necessary to break the chemical bonds in reactants and form new chemical bonds in the products. Catalysts bring reactants closer together in the appropriate orientation and weaken bonds, increasing the reaction rate. Without enzymes, chemical reactions would occur too slowly to sustain life.

The functionality of an enzyme is determined by the shape of the enzyme. The area in which bonds of the reactant(s) are broken is known as the active site. The reactants of enzyme catalyzed reactions are called substrates. The active site of an enzyme recognizes, confines, and orients the substrate in a particular direction.

Enzymes are substrate specific, meaning that they catalyze only specific reactions. For example, proteases (enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by the enzyme amylase). Notice that both of these enzymes end in the suffix -ase. This suffix indicates that a molecule is an enzyme.

Environmental factors may affect the ability of enzymes to function. You will design a set of experiments to examine the effects of temperature, pH, and substrate concentration on the ability of enzymes to catalyze chemical reactions. In particular, you will be examining the effects of these environmental factors on the ability of catalase to convert H 2 O 2 into H 2 O and O 2 .

The Scientific Method

As scientists, biologists apply the scientific method. Science is not simply a list of facts, but is an approach to understanding the world around us. It is use of the scientific method that differentiates science from other fields of study that attempt to improve our understanding of the world.

The scientific method is a systematic approach to problem solving. Although some argue that there is not one single scientific method, but a variety of methods; each of these approaches, whether explicit or not, tend to incorporate a few fundamental steps: observing, questioning, hypothesizing, predicting, testing, and interpreting results of the test. Sometimes the distinction between these steps is not always clear. This is particularly the case with hypotheses and predictions. But for our purposes, we will differentiate each of these steps in our applications of the scientific method.

You are already familiar with the steps of the scientific method from previous lab experiences. You will need to use your scientific method knowledge in today’s lab in creating hypotheses for each experiment, devising a protocol to test your hypothesis, and analyzing the results. Within the experimentation process it will be important to identify the independent variable, the dependent variable, and standardized variables for each experiment.

Part 1: Observe the Effects of Catalase

• Obtain two test tubes and label one as A and one as B.
• Use your ruler to measure and mark on each test tube 1 cm from the bottom.
• Fill each of two test tubes with catalase (from the potato) to the 1 cm mark
• Add 10 drops of hydrogen peroxide to the tube marked A.
• Add 10 drops of distilled water to the tube marked B.
• Bubbling height tube A
• Bubbling height tube B
• What happened when H 2 O 2 was added to the potato in test tube A?
• What caused this to happen?
• What happened in test tube B?
• What was the purpose of the water in tube B?

Part 2: Effects of pH, Temperature, and Substrate Concentration

Observations.

From the introduction and your reading, you have some background knowledge on enzyme structure and function. You also just observed the effects of catalase on the reaction in which hydrogen peroxide breaks down into water and oxygen.

From the objectives of this lab, our questions are as follows:

• How does temperature affect the ability of enzymes to catalyze chemical reactions?
• How does pH affect the ability of enzymes to catalyze chemical reactions?
• What is the effect of substrate concentration on the rate of enzyme catalyzed reactions?

Based on the questions above, come up with some possible hypotheses. These should be general, not specific, statements that are possible answers to your questions.

• Temperature hypothesis
• pH hypothesis
• Substrate concentration hypothesis

Test Your Hypotheses

Based on your hypotheses, design a set of experiments to test your hypotheses. Use your original experiment to shape your ideas. You have the following materials available:

• Catalase (from potato)
• Hydrogen peroxide
• Distilled water
• Hot plate (for boiling water)
• Acidic pH solution
• Basic pH solution
• Thermometer
• Ruler and wax pencil

Write your procedure to test each hypothesis. You should have three procedures, one for each hypothesis. Make sure your instructor checks your procedures before you continue.

• Procedure 1: Temperature
• Procedure 2: pH
• Procedure 3: Concentration

Record your results—you may want to draw tables. Also record any observations you make. Interpret your results to draw conclusions.

• Do your results match your hypothesis for each experiment?
• Do the results reject or fail to reject your hypothesis and why?
• What might explain your results? If your results are different from your hypothesis, why might they differ? If the results matched your predictions, hypothesize some mechanisms behind what you have observed.

Communicating Your Findings

Scientists generally communicate their research findings in written reports. Save the things that you have done above. You will be use them to write a lab report a little later in the course.

Sections of a Lab Report

• Title Page: The title describes the focus of the research. The title page should also include the student’s name, the lab instructor’s name, and the lab section.
• Introduction: The introduction provides the reader with background information about the problem and provides the rationale for conducting the research. The introduction should incorporate and cite outside sources. You should avoid using websites and encyclopedias for this background information. The introduction should start with more broad and general statements that frame the research and become more specific, clearly stating your hypotheses near the end.
• Methods: The methods section describes how the study was designed to test your hypotheses. This section should provide enough detail for someone to repeat your study. This section explains what you did. It should not be a bullet list of steps and materials used; nor should it read like a recipe that the reader is to follow. Typically this section is written in first person past tense in paragraph form since you conducted the experiment.
• Results: This section provides a written description of the data in paragraph form. What was the most reaction? The least reaction? This section should also include numbered graphs or tables with descriptive titles. The objective is to present the data, not interpret the data. Do not discuss why something occurred, just state what occurred.
• Discussion: In this section you interpret and critically evaluate your results. Generally, this section begins by reviewing your hypotheses and whether your data support your hypotheses. In describing conclusions that can be drawn from your research, it is important to include outside studies that help clarify your results. You should cite outside resources. What is most important about the research? What is the take-home message? The discussion section also includes ideas for further research and talks about potential sources of error. What could you improve if you conducted this experiment a second time?

Contributors and Attributions

• Biology 101 Labs. Authored by : Lynette Hauser. Provided by : Tidewater Community College. Located at : http://www.tcc.edu/ . License : CC BY: Attribution
• BIOL 160 - General Biology with Lab. Authored by : Scott Rollins. Provided by : Open Course Library. Located at : http://opencourselibrary.org/biol-160-general-biology-with-lab/ . License : CC BY: Attribution

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Introduction to enzymes, factors affecting enzyme activity.

Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to understand the basic enzymatic mechanism and to select a method for enzyme analysis. The conditions selected to measure the activity of an enzyme would not be the same as those selected to measure the concentration of its substrate. Several factors affect the rate at which enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators.

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Factors Affecting Enzyme Activity

Factor 1: concentration of enzyme, factor 2: concentration of substrate, factor 3: effect of temperature, factor 4: effect of ph, factor 5: effect of activators, which factors affect the activity of enzymes, how does enzyme concentration affect the activity of enzymes, how does ph affect the activity of enzymes, what happens to enzymes on very high temperatures, image source.

• EnzymesSave

Factors affecting enzyme activity include temperature, pH, substrate concentration, and the presence of inhibitors or activators. These ultimately affect the rate of chemical reactions in biological systems. Keep reading for more detailed A-level Biology revision!

Read more about Enzyme Inhibitors

• Biochemical reactions are necessary for growth, repairing damaged tissues, and obtaining energy and they take place in all living organisms’ bodies. These reactions are called ‘metabolism’ and they happen all the time in living organisms. If they stop working, this leads to the death of the organism.
• All the reactions that occur in living organisms require high activation energy to take place. To reduce the cell’s consumption of energy, there is a catalyst to ensure that the chemical reactions occur rapidly and reduce the activation of energy. This catalyst is the enzymes.
• Enzymes are biological catalysts made up of large protein molecules. They speed up the chemical reactions inside the cell. The enzyme is made up of a combination of amino acids which for a chain of polypeptides between each other.
• Enzymes are similar to other chemical catalysts. They participate in the reaction without getting affected. In other words, they speed up the chemical reactions inside the cells without getting consumed. Enzymes are affected by the hydrogen ion concentration (pH) and the temperature. Enzymes are highly specific compared to other catalysts, and each enzyme is specialized for one reactant substance. This reactant substance is called substrate, and it is specialized for one type of reaction or a few reactions. Enzymes lower the activation energy required to get the reaction started. Collectively, these are the most important properties of the enzyme.
• There are several factors that affect the speed of an enzyme’s action, such as the concentration of the enzyme, the concentration of the substrate, temperature, hydrogen ion concentration (pH), and the presence of inhibitors.
• As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. This property is used for determining the activities of serum enzymes during the diagnosis of diseases.
• In the presence of a given amount of enzyme, the rate of enzymatic reaction increases as the substrate concentration increases until a limiting rate is reached, after which further increase in the substrate concentration produces no significant change in the reaction rate. At this point, so much substrate is present that essentially all of the enzyme active sites have substrate bound to them.
• In other words, the enzyme molecules are saturated with substrate. The excess substrate molecules cannot react until the substrate already bound to the enzymes has reacted and been released (or been released without reacting).
• The protein nature of the enzymes makes them extremely sensitive to thermal changes. Enzyme activity occurs within a narrow range of temperatures compared to ordinary chemical reactions. As you have seen, each enzyme has a certain temperature at which it is more active. This point is called the optimal temperature, which ranges between 37 to 40C°.
• The enzyme activity gradually lowers as the temperature rises more than the optimal temperature until it reaches a certain temperature at which the enzyme activity stops completely due to the change of its natural composition.
• On the other hand, if the temperature lowers below the optimal temperature, the enzyme activity lowers until the enzyme reaches a minimum temperature at which the enzyme activity is the least. The enzyme activity stops completely at 0C°, but if the temperature rises again, then the enzyme gets reactivated once more.
• The potential of hydrogen (pH) is the best measurement for determining the concentration of hydrogen ion (H + )in a solution. It also determines whether the liquid is acidic, basic or neutral. Generally, all liquids with a pH below 7 are called acids, whereas liquids with a pH above 7 are called bases or alkalines. Liquids with pH 7 are neutral and equal the acidity of pure water at 25 C°. You can determine pH of any solution using the pH indicators.
• Enzymes are protein substances that contain acidic carboxylic groups (COOH – ) and basic amino groups (NH 2). So, the enzymes are affected by changing the pH value.
• Each enzyme has a pH value that it works at with maximum efficiency called the optimal pH. If the pH is lower or higher than the optimal pH, the enzyme activity decreases until it stops working. For example, pepsin works at a low pH, i.e, it is highly acidic, while trypsin works at a high pH, i.e, it is basic. Most enzymes work at neutral pH 7.4.
• Some of the enzymes require certain inorganic metallic cations, like Mg 2+ , Mn 2+ , Zn 2+ , Ca 2+ , Co 2+ , Cu 2+ , Na + , K + etc., for their optimum activity. Rarely, anions are also needed for enzyme activity, e.g. a chloride ion (CI – ) for amylase.

Frequently Asked Questions on Factors Affecting Enzyme Activity

The factors affecting the activity of enzymes are given below:

 Enzyme concentration

 Substrate concentration

 Temperature

The rate of reaction is directly proportional to the concentration of enzyme in that reaction. By increasing the enzymes concatenated, the rate of reaction can be increased.

Every enzyme works efficiently on optimum pH. A change in the optimum pH results in the denaturation of the enzyme.

The rate of reaction is increased with the increase of temperature but up to a certain limit. If the temperature is increased further, the enzyme is denatured.

Enzymes Save

Practical Biology

A collection of experiments that demonstrate biological concepts and processes.

Observing earthworm locomotion

Practical Work for Learning

Published experiments

Investigating an enzyme-controlled reaction: catalase and hydrogen peroxide concentration, class practical or demonstration.

Hydrogen peroxide ( H 2 O 2 ) is a by-product of respiration and is made in all living cells. Hydrogen peroxide is harmful and must be removed as soon as it is produced in the cell. Cells make the enzyme catalase to remove hydrogen peroxide.

This investigation looks at the rate of oxygen production by the catalase in pureed potato as the concentration of hydrogen peroxide varies. The oxygen produced in 30 seconds is collected over water. Then the rate of reaction is calculated.

Lesson organisation

You could run this investigation as a demonstration at two different concentrations, or with groups of students each working with a different concentration of hydrogen peroxide. Individual students may then have time to gather repeat data. Groups of three could work to collect results for 5 different concentrations and rotate the roles of apparatus manipulator, result reader and scribe. Collating and comparing class results allows students to look for anomalous and inconsistent data.

Apparatus and Chemicals

For each group of students:.

Pneumatic trough/ plastic bowl/ access to suitable sink of water

Conical flask, 100 cm 3 , 2

Syringe (2 cm 3 ) to fit the second hole of the rubber bung, 1

Measuring cylinder, 100 cm 3 , 1

Measuring cylinder, 50 cm 3 , 1

Clamp stand, boss and clamp, 2

Stopclock/ stopwatch

For the class – set up by technician/ teacher:

Hydrogen peroxide, range of concentrations, 10 vol, 15 vol, 20 vol, 25 vol, and 30 vol, 2 cm 3 per group of each concentration ( Note 1 )

Pureed potato, fresh, in beaker with syringe to measure at least 20 cm 3 , 20 cm 3 per group per concentration of peroxide investigated ( Note 2 )

Rubber bung, 2-holed, to fit 100 cm 3 conical flasks – delivery tube in one hole (connected to 50 cm rubber tubing)

Health & Safety and Technical notes

Wear eye protection and cover clothing when handling hydrogen peroxide. Wash splashes of pureed potato or peroxide off the skin immediately. Be aware of pressure building up if reaction vessels become blocked. Take care inserting the bung in the conical flask – it needs to be a tight fit, so push and twist the bung in with care.

Read our standard health & safety guidance

1 Hydrogen peroxide: (See CLEAPSS Hazcard) Solutions less than 18 vol are LOW HAZARD. Solutions at concentrations of 18-28 vol are IRRITANT. Take care when removing the cap of the reagent bottle, as gas pressure may have built up inside. Dilute immediately before use and put in a clean brown bottle, because dilution also dilutes the decomposition inhibitor. Keep in brown bottles because hydrogen peroxide degrades faster in the light. Discard all unused solution. Do not return solution to stock bottles, because contaminants may cause decomposition and the stock bottle may explode after a time.

2 Pureed potato may irritate some people’s skin. Make fresh for each lesson, because catalase activity reduces noticeably over 2/3 hours. You might need to add water to make it less viscous and easier to use. Discs of potato react too slowly.

3 If the bubbles from the rubber tubing are too big, insert a glass pipette or glass tubing into the end of the rubber tube.

SAFETY: Wear eye protection and protect clothing from hydrogen peroxide. Rinse splashes of peroxide and pureed potato off the skin as quickly as possible.

Preparation

a Make just enough diluted hydrogen peroxide just before the lesson. Set out in brown bottles ( Note 1 ).

b Make pureed potato fresh for each lesson ( Note 2 ).

c Make up 2-holed bungs as described in apparatus list and in diagram.

Investigation

d Use the large syringe to measure 20 cm 3 pureed potato into the conical flask.

e Put the bung securely in the flask – twist and push carefully.

f Half-fill the trough, bowl or sink with water.

g Fill the 50 cm 3 measuring cylinder with water. Invert it over the trough of water, with the open end under the surface of the water in the bowl, and with the end of the rubber tubing in the measuring cylinder. Clamp in place.

h Measure 2 cm 3 of hydrogen peroxide into the 2 cm 3 syringe. Put the syringe in place in the bung of the flask, but do not push the plunger straight away.

i Check the rubber tube is safely in the measuring cylinder. Push the plunger on the syringe and immediately start the stopclock.

j After 30 seconds, note the volume of oxygen in the measuring cylinder in a suitable table of results. ( Note 3 .)

k Empty and rinse the conical flask. Measure another 20 cm 3 pureed potato into it. Reassemble the apparatus, refill the measuring cylinder, and repeat from g to j with another concentration of hydrogen peroxide. Use a 100 cm 3 measuring cylinder for concentrations of hydrogen peroxide over 20 vol.

l Calculate the rate of oxygen production in cm 3 /s.

m Plot a graph of rate of oxygen production against concentration of hydrogen peroxide.

Teaching notes

Note the units for measuring the concentration of hydrogen peroxide – these are not SI units. 10 vol hydrogen peroxide will produce 10 cm 3 of oxygen from every cm 3 that decomposes.( Note 1 .)

In this procedure, 2 cm 3 of 10 vol hydrogen peroxide will release 20 cm 3 of oxygen if the reaction goes to completion. 2 cm 3 of liquid are added to the flask each time. So if the apparatus is free of leaks, 22 cm 3 of water should be displaced in the measuring cylinder with 10 vol hydrogen peroxide. Oxygen is soluble in water, but dissolves only slowly in water at normal room temperatures.

Use this information as a check on the practical set-up. Values below 22 cm 3 show that oxygen has escaped, or the hydrogen peroxide has not fully reacted, or the hydrogen peroxide concentration is not as expected. Ask students to explain how values over 22 cm 3 could happen.

An error of ± 0.05 cm 3 in measuring out 30 vol hydrogen peroxide could make an error of ± 1.5 cm 3 in oxygen production.

Liver also contains catalase, but handling offal is more controversial with students and introduces a greater hygiene risk. Also, the reaction is so vigorous that bubbles of mixture can carry pieces of liver into the delivery tube.

If collecting the gas over water is complicated, and you have access to a 100 cm 3 gas syringe, you could collect the gas in that instead. Be sure to clamp the gas syringe securely but carefully.

The reaction is exothermic. Students may notice the heat if they put their hands on the conical flask. How will this affect the results?

Health and safety checked, September 2008

http://www.saps.org.uk/secondary/teaching-resources/293-student-sheet-24-microscale-investigations-with-catalase Microscale investigations with catalase – which has been transcribed onto this site at Investigating catalase activity in different plant tissues.

(Website accessed October 2011)

Explain the factors affecting enzyme activity.

Enzymes are biological catalysts that are proteinaceous in nature. the factors affecting the enzyme activity are listed below: 1. substrate concentration: the activity of an enzyme also increases with the increase in substrate concentration. if the substrate concentration increases, then the availability of the active site would decrease. this will affect the activity of an enzyme and limit the reaction rate. 2. ph each enzyme has its optimal ph in which they work. for example pepsin and trypsin work on acidic ph. the enzymes are globular proteinaceous structure, form by the interaction of the hydrogen bond between the side chains of the protein. any change in the cause deionization of side chain which results in the denaturation of the enzyme. 3. temperature: each enzyme works on its optimal temperature. any alteration in temperature affects the activity of an enzyme, and it also leads to denaturation of an enzyme. 4. enzyme cofactor and coenzyme: each enzyme requires cofactors (inorganic ion or protein organic molecules) for their work. the non-availability of these cofactors decreases the activity of an enzyme. 5. enzyme inhibitors: the inhibitors of an enzyme bind to the active site which affects the activity of an enzyme..