Definition of a Katal (unit of enzyme activity)

Definition of a Katal (unit of enzyme activity)

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I am very confused about what one 'Katal' actually is. From Wikipedia,

"The katal is not used to express the rate of a reaction; that is expressed in units of concentration per second (or moles per liter per second). Rather, it is used to express catalytic activity which is a property of the catalyst. The katal is invariant of the measurement procedure, but the numerical quantity value is not and depends on the experimental conditions. Therefore, in order to define the quantity of a catalyst, the rate of conversion of a defined chemical reaction is specified as mols reacted per second. One katal of trypsin, for example, is that amount of trypsin which breaks a mole of peptide bonds per second under specified conditions."

So if a Katal is a specific AMOUNT of trypsin, then why is it expressed in units of moles per second. This is not dimensionally consistent! An 'amount' could be expressed as moles or mass, but certainly not as moles per second which is like a rate of reaction multiplied by volume… ? The units and definition just seem to be all wrong.

If I were to take it that the Katal is the amount of enzyme catalysing one mole of a reaction per second, then this would mean that an enzyme with a smaller number of Katals of activity would actually be more active because less of it is required to catalyse one mole of the reaction per second?

A katal is indeed a specific amount of enzyme (rather than a reaction rate), but one which is measured with respect to the catalytic activity of the enzyme, rather than with respect to the number of molecules, their weight, or their size.

Roughly speaking, one katal is the amount of of enzyme which can convert 1 mole in 1 second. (Just like one pascal is the amount of pressure which applies one newton of force to an area of one square meter.) Or to rephrase things slightly, just like you can take an object of a particular length and express that length as "X meters" (being the number of meter sticks you can line up next to that length), you can take a given amount of enzyme and express the catalytic activity of that quantity of enzyme as "X katals", being the number of moles the enzyme can convert in one second.

This is not an intrinsic property of the enzyme itself. (It's an "extensive", not an "intensive" property.) The number of katals varies based on how much enzyme you have - if you have twice the amount of enzyme, you have twice the number of katals. (Just like you have twice the number of grams or twice the number of moles.)

As such, you can't talk about "an enzyme with a smaller number of katals of activity". I can understand the confusion, though, given the use of "activity" in the name. A paper the Wikipedia article references talks about the fact that there was some argument for calling the quantity being measured 'catalytic amount' rather than 'catalytic activity'

As to why measuring enzyme amounts in katals versus moles or grams, the argument is similar to why there are separate units for mass (gram) and number (mole). It expresses a feature of the "size" of the enzyme not captured by the other units.

Although this is old news, as @R.M. has been good enough to provide an answer, I feel I should add my own. The reason is not that I think his answer is incorrect in any way - it is technically quite correct and I am not going to repeat or rephrase his technical arguments - but I do not think it addresses what I regard as the misunderstanding in the poster's argument - a misunderstanding that others might make, even if the OP has progressed to other things.

The problem is one of semantics - What do you understand by the word 'amount'?

The OP is clear. She writes:

“An 'amount' could be expressed as moles or mass, but certainly not as moles per second… ”

So her understanding of the word amount is tied to the (extrinsic) mass property of an enzyme, which she clearly feels is intimately related to the number of molecules.

It seems to me that the way to deal with this argument is to point out that it is a misconception - there are properties of molecules other than mass that can be, and are, used to quantify them, and for enzymes catalytic activity is one such property, and the one that must be used in practice.

In practice, quantitation of molecules (the process that produces an amount) has to be performed in terms of their measurable properties when we are unable to 'count their number' (as it were). Mass is actually indirect - to be of use we need to know the purity of the compound and its molecular weight. Likewise we may quantify molecules by e.g. their light absorbance at a particuar wavelength, but to do this we need a molar reference value which will differ, e.g. between NADH and the amino acid tryptophan. Quantification using enzyme activity may be thought of in this light.

What is the Difference Between Enzyme Activity and Specific Activity

The main difference between enzyme activity and specific activity is that enzyme activity is the moles of substrate converted by the enzyme per unit time whereas specific activity is the activity of enzyme per milligram of total enzyme. Furthermore, enzyme activity measures the amount of active enzymes present under a given condition while specific activity measures the enzyme purity in the mixture.

Enzyme activity and specific activity are two enzyme units which measure the enzymatic activity. The measurement of enzymatic activity by enzyme assays is important for the study of enzyme kinetics as well as enzyme inhibition.

Key Areas Covered

Key Terms

Enzyme Activity, Enzyme Purity, Enzyme Units, Specific Activity, Substrate Concentration

Expression and Measurement of Enzyme Activity

The presence or absence of an enzyme is typically determined by observing the rate of the reaction(s) it catalyzes. Quantitative enzyme assays are designed to measure either the total amount of a particular enzyme (or class of enzymes) in units of moles or, more commonly, the catalytic activity associated with a particular enzyme. The two types of assays differ in that those in the latter category measure only active enzyme. The assays contained in this section are concerned primarily with the measurement of catalytic activity, or active enzyme. The assays are based on kinetic experiments, as activities are calculated from measured reaction rates under defined conditions. The basic Premise for these assays is that the amount of enzyme in a reaction mixture can be determined from the rate at which the enzyme-catalyzed reaction occurs.


The goal of most enzyme assays is to quantitatively measure the amount of enzyme activity (catalytic activity) present in a sample. Thus, assay results are typically reported in "activity" units. A unit of activity may be defined in various ways, but all such units are ultimately based on rates of substrate consumption and/or product formation. The most common units for expressing catalytic activity are the International Unit (also called the Unit or Enzyme Unit U) and the Katal (Kat). The International Union of Biochemistry (IUB) originally defined a standard unit of enzyme activity (1 U) as that amount of enzyme that catalyzes the formation of 1 |mol product (or the conversion of 1 |imol substrate) per minute under standard conditions. In 1979, the Nomenclature Committee of the IUB recommended the use of the Katal as the fundamental unit of enzyme activity. The Katal is defined as that amount of enzyme that catalyzes the formation of 1 mol product (or the conversion of 1 mol substrate) per second under defined conditions. Thus, 1 Kat is equivalent to 6 x 107 U. The Katal was recommended because it is consistent with Système International (SI Units).

It is also common for enzyme activities to be reported in units based on changes in reaction mixture properties that are themselves a function of the extent of the enzymatic reaction. These units are often difficult to interpret in

Substrate concentration [S]

Figure C1.1.1 Relationship between substrate concentration and initial velocity at fixed enzyme concentrations. The Michaelis constant, KM, is equal to the substrate concentration corresponding to one-half Vmax.

Figure C1.1.1 Relationship between substrate concentration and initial velocity at fixed enzyme concentrations. The Michaelis constant, KM, is equal to the substrate concentration corresponding to one-half Vmax.

Strategies for Enzyme Activity Measurements

Figure C1.1.2 Time course of product generation for typical enzyme-catalyzed reaction. Product concentration is shown to asymptotically approach its equilibrium value (horizontal dashed line). The diagonal dashed line illustrates the portion of the curve used to calculate initial velocity.

Expression and Measurement of Enzyme Activity terms of absolute numbers of catalytic events, but they can be used to effectively communicate relative amounts of enzyme activity. Assays of this type are particularly common when complex substrates and/or heterogeneous reaction mixtures are used. Changes in solubility, turbidity, and viscosity, per unit time, are examples of such units.

The upshot of the above discussion is that a variety of enzyme activity units may be encoun -tered. This makes it essential that all units, whatever their bases, be clearly defined.


Enzyme assays are typically done under relevant conditions, be they physiological conditions, food-storage conditions, or conditions corresponding to maximal activity. This implies that consideration must be given to reaction mixture parameters such as pH, temperature, ionic strength, buffer composition, and other components not involved in the reaction. It is prudent to assume that changes in any of these parameters may affect enzyme activity. Analysts will often run assays under apparent optimum conditions (maximal activity), such as optimum pH, because these conditions tend to coincide with maximum assay sensitivity. It should be apparent from this discussion that assays using different reaction conditions may have only limited comparative value.

Substrate concentration is yet another variable that must be clearly defined. The hyperbolic relationship between substrate concentration ([S]) and reaction velocity, for simple enzyme-based systems, is well known (Figure C1.1.1). At very low substrate concentrations ([S]<<Km), there is a linear first-order dependence of reaction velocity on substrate concentration. At very high substrate concentrations ([S]>>^M), the reaction velocity is essentially independent of substrate concentration. Reaction velocities at intermediate substrate concentrations ([S]

^M) are mixed-order with respect to the concentration of substrate. If an assay is based on initial velocity measurements, then the defined substrate concentration may fall within any of these ranges and still provide a quantitative estimate of total enzyme activity (see Equation C1.1.5). The essential point is that a single substrate concentration must be used for all calibration and test-sample assays. In most cases, assays are designed such that [S]>>^M, where small deviations in substrate concentration will have a minimal effect on reaction rate, and where accurate initial velocity measurements are typically easier to obtain.

The composition of assay reaction mixtures is generally defined to the extent that it is practical. In most cases, the major source of unknowns is the enzyme preparation itself. Ki-netically relevant compounds endogenous to the enzyme source, such as substrates, inhibitors, and activators, may partition with the enzyme during sample preparation. Hence, for comparative purposes, it is recommended that enzyme preparation schemes be standardized.


It is generally assumed that reported enzyme activities are based on initial velocity experiments, and that reported activities are proportional to the amount of active enzyme in the reaction mixture. Analysts should independently verify these assumptions in their laboratories, using the experimental systems particular to their situations. Initial velocity (or initial rate) kinetics implies that one is measuring the instantaneous velocity at the substrate concentration corresponding to the initiation of the reaction. Realistically, initial velocity often means the velocity that is measured as close to the initiation of the reaction as possible. Initial velocities are typically determined by collecting data points immediately after the initiation of the reaction, analyzing the data to confirm that the initial rate was actually observed (this may be as simple as showing that early data points define a linear rate of product formation), and then calculating the corresponding velocity in appropriate units. If the reaction is such that it is not experimentally feasible to obtain an initial linear rate of product formation, then a nonlinear regression technique can be used to estimate the initial velocity from the early phase of the reaction's time course.

Figure C1.1.2 shows a typical time course resulting from a continuous assay of product formation in an enzyme-catalyzed reaction. The hyperbolic nature of the curve illustrates that the reaction rate decreases as the reaction nears completion. The reaction rate, at any given time, is the slope of the line tangent to the curve at the point corresponding to the time of interest. Reaction rates decrease as reactions progress for several reasons, including substrate depletion, reactant concentrations approaching equilibrium values (i.e., the reverse reaction becomes relevant), product inhibition, enzyme inactivation, and/or a change in reaction conditions (e.g., pH as the reaction proceeds). With respect to each of these reasons, their effects will be at a minimum in the initial phase of the reaction—i.e., under conditions corresponding to initial velocity measurements. Hence, the interpretation of initial velocity data is relatively simple and thus widely used in enzyme-related assays.


Enzyme assays are typically designed with the presumption that measured activities will be directly proportional to the amount of active enzyme in the reaction mixtures. Thus, the conceptual basis for most assays is that a linear relationship exists between measured activity and catalytic potential (active enzyme). This simple relationship is unlikely to be valid for all experimental permutations, and thus tests verifying the relationship between measured activity and quantity of enzyme should be included in all assays. This can be accomplished in a relatively easy way by assaying a series of samples that cover a range of enzyme concentrations and, based on the data, preparing a calibration curve of measured activity versus (relative) enzyme concentration, while making sure that enzyme concentrations in actual test samples fall within this range. For reference, enzyme concentrations in traditional assays are typically in the nanomolar to micromolar range.

A single-substrate reaction can be depicted as

where E is enzyme S is substrate ES is enzyme-substrate complex P is product and ki, k2, and k3 are rate constants. The kinetics of such a unireactant enzyme system are most often described by the basic equation of enzyme kinetics, the Michaelis-Menten equation.

It expresses the velocity (v) of a single-substrate reaction (Equation C1.1.1) in terms of substrate concentration at time zero ([S]) and the kinetic constants KM and Vmax. Vmax is defined as the limiting maximal velocity for the reaction, which is observed when all of the enzyme is present as ES. KM, known as the Michaelis constant, is a pseudoequilibrium constant, which equals the concentration of substrate at which the reaction velocity equals one-half Vmax (Figure C1.1.1). Strategies for

Enzyme Activity Measurements

Vmax and KM are defined by the following equations:

The velocity term, v, in Equation C 1.1.2 refers to measured initial velocities. The equation's derivation is based on the assumptions that enzyme concentrations are much less than substrate concentrations ([E]total<<[S]) and that, over the course of the assay, the concentration of the enzyme-substrate complex remains essentially constant (the steady-state assumption). The point of this discussion is to show that measured initial velocities are expected to be directly proportional to the amount of active enzyme in reaction mixtures. Equation C1.1.5, obtained by substituting and rearranging the equations above, more clearly illustrates this relationship.

In this equation, note that bracketed terms are constants under initial velocity conditions.

A nonlinear relationship between enzyme concentration and measured activity is indicative of a more complex reaction system. Complications of this nature may arise from such things as changes in the composition of the reaction mixture (e.g., pH due to the addition of increasing amounts of enzyme solution), assay limitations (e.g., insufficient substrate), limited coupling-enzyme (where assays are based on coupled enzyme systems), the presence of inhibitors, and enzyme-cofactor or enzyme-enzyme dissociation phenomena. Nonlinear relationships may also be an inherent outcome of assays employing complex substrates, such as starch granules, cellulose, and protein networks. In all cases it is advisable to verify the nature of the relationship between measured activity and quantity of enzyme (as discussed earlier). If the relationship is found to be nonlinear, then it is prudent to carefully check the assay to make sure that the nonlinear relationship is not simply an artifact of the experimental method.

Eisenthal, R. and Danson, M.J. (eds.) 1992. Enzyme Assays: A Practical Approach. IRL Press, Oxford.

A good source of information on the design and execution of enzyme assays. The initial chapter, by K.F. Tipton, provides an excellent discussion of the general principles involved in enzyme assays. Subsequent chapters deal with specific assay approaches.

Segel, I.H. 1975. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley & Sons, New York.

A detailed, yet readable, discussion of rapid equilibrium and steady-state kinetics. The initial portion of the book deals with basic enzyme biochemistry, the kinetics of simple unireactant enzymes, and simple inhibition systems.

Whitaker, J.R. 1994. Principles of Enzymology for the Food Sciences, 2nd ed. Marcel Dekker, New York.

A classic text used for nearly 30 years to teach undergraduate and graduate students in the food sciences the fundamental principles of enzymology. The latter part of the text emphasizes the biochemistry of enzymes of particular relevance to the food

Wong, D.W.S. 1995. Food Enzymes: Structure and Mechanism. Chapman and Hall, New York.

This book gives an informative, yet relatively concise and well-referenced, summary of the structure and catalytic mechanism of selected food-related enzymes. All enzymes coveredplay an important role in food systems and all have their catalytic mechanism described at the molecular level.

Contributed by Michael H. Penner Oregon State University Corvallis, Oregon k


All enzymes are globular proteins with a specific tertiary structure, which catalyse metabolic reactions in all living organisms. This means that they speed up chemical reactions, but are not ‘used-up’ as part of the reaction.

Enzymes are relatively large molecules, consisting of hundreds of amino acids which are responsible for maintaining the specific tertiary structure of the enzyme. Each enzyme has a specific active site shape, maintained by the specific overall tertiary structure. Therefore the tertiary structure must not be changed.

(b) state that enzyme action may be intracellular or extracellular

Extracellular enzyme action occurs outside the cell, which produces the protein. For example, some enzymes in digestive systems are extracellular as they are released from the cells that make them, onto food within the digestive system spaces.

Intracellular enzyme action occurs inside the cell, which produces the enzyme. For example, some enzymes in digestive systems are found in the cytoplasm of cells or attached to cell membranes and the reaction takes place inside the cell.

(c) describe, with the aid of diagrams, the mechanism of action of enzyme molecules, with reference to specificity, active site, lock and key hypothesis, induced-fit hypothesis, enzyme-substrate complex, enzyme-product complex and lowering of activation energy.

The activation energy is the minimum level of energy required to enable a reaction to take place. Enzymes work by lowering the activation energy of reactions. This means reactions can proceed quickly at temperatures much lower than boiling point as less energy is required for the reaction.

(d) describe and explain the effects of pH, temperature, enzyme concentration and substrate concentration on enzyme activity

(e) describe how the effects of pH, temperature, enzyme concentration and substrate concentration on enzyme activity can be investigated experimentally

(f) explain the effects of competitive and non-competitive inhibitors on the rate of enzyme-controlled reactions, with reference to both reversible and non-reversible inhibitors

An enzyme inhibitor is any substance or molecule that slows down the rate of an enzyme-controlled reaction by affecting the enzyme molecule is some way.

Reversible inhibitors are inhibitors that bind to the active site for a short period and then leave. The removal of the inhibitor from the reacting mixture leaves the enzyme molecules unaffected.

Irreversible inhibitors are inhibitors that bind permanently to the enzyme molecule. Any enzyme molecules bound by inhibitor molecules are effectively denatured.

(g) explain the importance of cofactors and coenzymes in enzyme controlled reactions

A cofactor is any substance that must be present to ensure enzyme-controlled reactions can take place at the appropriate rate. Some cofactors are part of the enzymes (prosthetic groups) others affect the enzyme on a temporary basis (coenzymes and inorganic ion cofactors).

(h) state that metabolic poisons may be enzyme inhibitors, and describe the action of one named poison

(i) state that some medicinal drugs work by inhibiting the activity of enzymes

External links

  • Unit "katal" for catalytic activity (IUPAC Technical Report) Pure Appl. Chem. Vol. 73, No. 6, pp.   927� (2001)
  • René Dybkær (1 March 2002). "The Tortuous Road to the Adoption of katal for the Expression of Catalytic Activity by the General Conference on Weights and Measures". Clinical Chemistry. 48 (3): 586�. doi: 10.1093/clinchem/48.3.586 . PMID   11861460. Archived from the original on 6 October 2008 . Retrieved 8 December 2005 .
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Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

Titrimetric Stop Reaction

0.5%(w/v) Polygalacturonic Acid (Sub)
Prepare by adding 200 mL of purified water to 1.00 g to 1.05 g of polygalcturonic acid (Product No. P3889) with stirring.
With stirring, bring to a boil and maintain boiling for exactly five minutes. Immediately place in a 25 ºC water bath with an immersible stirrer. Stir until the temperature is at 25 ºC and allow the solution to stir for an additional ten minutes.
With continued stirring, adjust the pH of the solution by adding 0.05 mL aliquots of Reagent 7.3.6 (NaOH-2) at 1 minute intervals until a pH of 4.0 is obtained. Add 0.05 mL aliquots of 2 N NaOH with stirring until a pH of 4.00 to 4.05 is maintained for a minimum of ten minutes. If particles are present, then filter through an Evergreen Column, maximum 90 μM frit.
Recheck pH and adjust with Reagent 7.3.3 (NaOH) at 25 ºC to 4.00 - 4.02 with stirring. The pH should remain in the range of 4.00 - 4.02 for a minimum of 30 minutes.
It may be necessary to recheck pH every fifteen minutes to ensure that the pH is within the range of 4.00 to 4.02 at 25 ºC. If not, adjust pH with 0.1N NaOH.

100 mM Iodine (I2)
Use Iodine volumetric Standard, 0.1 N (Product No. 318981).

1.0 N Sodium Hydroxide (NaOH) Solution (Product No. S2567).

1 M Sodium Carbonate (Na2CO3)
Prepare in purifed water at 106 mg/mL using Sodium Carbonate, Reagent Grade (Product No. S2127).

2.0 N Sulfuric Acid (H2SO4)
Prepare in purified water at 0.06 mL/mL using Sulfuric Acid, ACS Reagent (Product No. 258105).

10 N Sodium Hydroxide (NaOH-2)
Prepare at 400 mg/mL in purified water using Sodium Hydroxide, Reagent Grade (Product No. S5881).

100 mM Sodium Thiosulfate, Standardized (Na2S2O3)
Prepare in 3 L of purifed water using 78.3 g of Sodium Thiosulfate, Pentahydrate (Product No. S8503) and 0.6 g of Sodium Carbonate, Monohydrate (Product No. S4132). Standardize against a standard solution of potassium dichromate, prepared from potassium dichromate, NIST.

Pectinase Solution (Enzyme)
Immediately before use, prepare a solution containing approximately 100 unit/mL in cold purified water. Dissolution may require more than one minute of swirling and some insoluble particulates may still be present.

1.0 %(w/v) Starch (Starch)
Prepare in purified water at 10 mg/mL by boiling for exactly five minutes with stirring. Cool to room temperature. Use starch, potato soluble (Product No. S2004).

Pipette (in milliliters) the following reagents into suitable glass vessels in triplicate for control and/or sample:

Enzyme Activity Review in 26 Easy Questions

Catalysts are substances that reduce the activation energy of a chemical reaction, facilitating it or making it energetically viable. The catalyst increases the speed of the chemical reaction.

More Bite-Sized Q&As Below

2. What amount of catalyst is consumed in the reaction it catalyzes?

Catalysts are not consumed in the reactions they catalyze.

3. Is there a difference between the initial and the final energy levels in catalyzed and non-catalyzed reactions?

The catalysis does not alter the state of the energy of the reagents and products of a chemical reaction. Only the energy necessary for the reaction to occur, that is, the activation energy, is altered.

4. What are enzymes? What is the importance of enzymes for living beings?

Enzymes are proteins that are catalysts of chemical reactions. Chemistry shows us that catalysts are non-consumable substances that reduce the activation energy necessary for a chemical reaction to occur.

Enzymes are highly specific to the reactions they catalyze. They are of vital importance for life because most of the chemical reactions in cells and tissues are catalyzed by enzymes. Without enzyme action, those reactions would not occur or would not happen with the required speed for the biological processes in which they are involved.

The Enzyme-Substrate Complex

5. What are substrates of enzymatic reactions?

Substrates are reagent molecules upon which enzymes act.

Enzymes have spatial binding sites to attach to their substrate. These sites are called the activation centers of the enzyme. Substrates bind to these centers, forming the enzyme-substrate complex.

6. What are the main theoretical models that try to explain the formation of the enzyme-substrate complex?

There are two main models that explain the formation of the enzyme-substrate complex: the lock and key model and the induced fit model.

In the lock and key model, the enzyme has a region with a specific spatial conformation for the binding of the substrate. In the induced fit model, the binding of the substrate induces a change in the spatial configuration of the enzyme to make the substrate fit.

7. How does the formation of the enzyme-substrate complex explain the reduction in the activation energy of chemical reactions?

The enzyme possibly works as like a test tube within which reagents meet to form products. Enzymes facilitate this meeting, making it easier for collisions between reagents to occur and, as a result, the activation energy of the chemical reaction is reduced. This is one possible hypothesis.

8. On what structural level of the enzyme (primary, secondary, tertiary or quaternary) does the enzyme-substrate interaction depend?

The substrate binds to the enzyme at the activation centers. These are specific three-dimensional sites and therefore they depend on the protein's tertiary and quaternary structures. The primary and secondary structures, however, condition the other structures, and consequently are equally important.

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Specificity of Enzymatic Action

9. What is the activation center of an enzyme? Is it the key or the lock in the lock and key model?

The activation center is a region of the enzyme produced by its spatial conformation to which the substrate binds. In the lock and key model, the activation center is the lock and the substrate is the key.

10. Why enzyme action is considered highly specific?

Enzyme action is highly specific because only the specific substrates of an enzyme bind to the activation center of that enzyme. Each enzyme generally catalyzes only one specific chemical reaction.

Factors that Change Enzyme Activity

11. What happens to the functionality of a denatured enzyme? How can that result be explained with the help of the lock and key model?

According to the lock and key, enzyme functionality depends entirely on the integrity of the activation center, a molecular region with specific spatial characteristics. After denaturation, the spatial conformation of the protein is modified, the activation center is destroyed and the enzyme loses its catalytic activity.

12. What are the main factors that alter the speed of enzymatic reactions?

The main factors that change the speed of enzymatic reactions are temperature, pH and substrate concentration (quantity).

13. How does substrate concentration affect the speed of enzymatic reactions?

Initially, as substrate concentration increases, the speed of the reaction increases. This happens because free activation centers of the enzyme bind to free substrates. Once all the activation centers of the available enzymes are bound to their substrates, new increases in the substrate concentration will have no effect on the speed of the reaction.

14. How does temperature affect the action of enzymes on their substrates?

There are defined temperature ranges under which enzymes operate and there is a specific temperature level (optimum temperature) in which enzymes have maximum efficiency. Therefore, temperature variations affect enzyme activity and the speed of the reactions they catalyze.

In addition, because they are proteins, enzymes can be denatured under extreme temperatures.

15. Concerning enzymatic reactions, how different are the curves of the graph of the variation in the speed of a reaction as function of substrate concentration and the graph of the variation in the speed of a reaction as function of temperature?

The curve of the variation in speed of the enzymatic reaction as a function of increasing substrate concentration increases in a curve formation until approaching the point where it stabilizes due to the saturation of the activationꃎnters of the enzymes.

The curve of the variation in the speed of the enzymatic reaction as a function of increasing temperature initially increases and then reaches a peak (the optimum temperature), after which it decreases to zero at the point in which the enzymes are rendered inactive by denaturation.

16. What is the relationship between  the cooling of organs and tissues for medical transplants and the effect of temperature on enzymatic reactions?

Molecular degradation during the decomposition of organs and tissues is catalyzed by enzymes. The cooling to adequate temperatures of some organs and tissues destined for transplantation reduces that enzyme activity and thus decreases the natural decomposition process. By the same rationale, the cooling reduces the metabolic work of cells and prevents the breakdown of their own structures to obtain energy. A subsequent increase in temperature reverts the denaturation of enzymes, allowing the organs and tissues also preserved by other specific techniques to be grafted into the receptors.

17. Does pH affect enzyme activity?

The concentration of hydrogen ions in a solution affects enzyme activity. Each enzyme has a maximum efficiency in an optimum pH.

Since pH is one of the factors in the denaturation of proteins, if an enzyme is subject to a pH level under which it is denatured, there will be no enzymatic activity.

18. Do enzymes act better under acidic or alkaline pHs?

Most enzymes act under pHs between 6 and 8, a range that corresponds to the general acidic level of cells and blood. There are enzymes, however, that act only under very acid or very alkaline pH. Therefore, enzyme activity depends on pH range.

In the stomach, for example, gastric juice has a very low pH, around 2. Nonetheless, the enzyme pepsin acts to intensively digest proteins. In the duodenum, pancreatic secretions increase the pH of the intestinal juice to allow other digestive enzymes, such as trypsin, to act.

19. Since pepsin is a gastric enzyme, does it have an acidic or alkaline optimum pH? What happens to pepsin when it enters the duodenum?

Pepsin acts within the stomach so its optimum pH is around 2, an acidic pH. When the enzyme enters the duodenum, it comes in contact with a higher pH and its enzyme activity comes to and end.


20. What are enzyme cofactors?

Some enzymes need other associated molecules to work. These molecules are called enzyme cofactors and they can be organic ions like mineral salts, or organic molecules, to give some examples.

Inactive enzymes which are not bound to their cofactors are called apoenzymes. Active enzymes bound to their cofactors are called holoenzymes.

21. What is the relationship between vitamins and enzyme cofactors?

Many vitamins are enzyme cofactors that cannot be synthesized by the body and, as a result, must be obtained from the diet.

Enzyme Inhibitors, Allosterism and Zymogens

22. In a enzymatic reaction, what is the effect of a substance with the same spatial conformation as the enzyme substrate? How is this type of substance recognized?

Substances that “simulate” substrates can bind to the activation center of enzymes, thus blocking the true substrates from binding to these enzymes and paralyzing the enzymatic reaction. These “fake substrates” are called enzyme inhibitors.

The binding of enzyme inhibitors to enzymes can be reversible or irreversible.

Many medical drugs, including some antibiotics, antivirals, antineoplastics, antihypertensives and even sildenafil (trade name Viagra), are enzyme inhibitors that block enzyme activity.

23. What is the mechanism of action of the antibiotic penicillin?

Penicillin, discovered by the Scottish doctor Alexander Fleming in 1928, is a drug that inhibits the enzymes necessary for the synthesis of peptidoglycans, a component of the bacterial cell wall. Through this, the inhibition the bacterial population stops growing because there is no new cell wall formation.

Fleming won the Nobel Prize in medicine for the discovery of penicillin.

24. What is the mechanism of action of the antiretroviral drugs called protease inhibitors which are used against HIV infection?

Protease inhibitors are some of the antiretroviral drugs used to treat HIV infection. Protease is an enzyme necessary for the construction of the  human immunodeficiency virus (HIV)ꂯter the synthesis of its proteins within the host cell. The protease inhibitor binds to the activation center of the enzyme blocking the formation of the enzyme-substrate complex and enzyme activity, thus stopping viral replication.

25. What are allosteric enzymes?

Allosteric enzymes are enzymes with more than one activation center and to which other substances, called allosteric regulators, bind.

Allosteric regulators can be allosteric inhibitors or allosteric activators. The interaction between an allosteric enzyme and an allosteric inhibitor prohibits the binding of the substrate to the enzyme. The interaction between an allosteric enzyme and an allosteric activator allows the binding of the substrate to the enzyme and sometimes increases the affinity of the enzyme for the substrate. This regulatory phenomenon of enzyme activity is called allosterism.

26. What are zymogens?

Zymogens, or proenzymes, are enzymes secreted in inactive form. Under certain conditions, a zymogen changes into the active form of the enzyme. In general, zymogen secretions happen because enzyme activity can harm secretory tissue.

For example, the pepsinogen secreted by the stomach becomes active under an acidic pH, turning into the enzyme pepsin. Other well-known zymogens are trypsinogen and chymotrypsinogen, enzymes that are secreted by the exocrine pancreas and which become trypsin and chymotrypsin respectively.

The term allosterism refers to the fact that the activity of certain enzymes can be affected by the binding of small molecules. Molecules causing allosteric effects come in two classifications. Ones that are substrates for the enzymes they affect are called homotropic effectors and those that are not substrates are called heterotropic effectors.

The homotropic effectors usually are activators of the enzymes they bind to and the results of their action can be seen in the conversion of the hyperbolic curve typical of a V0 vs. [S] plot for an enzyme (Figure 4.18), being converted to a sigmoidal plot (Figure 4.44). This is due to the conversion of the enzyme from the T-state to the R-state on binding the substrate/homotropic effector.

Figure 4.44 - Kinetic profile of an allosteric enzyme whose activity is controlled by a homotropic effector. Image by Aleia Kim

The V0 vs. [S] plot of allosteric enzyme reactions resembles the oxygen binding curve of hemoglobin (see Figure 2.83). Even though hemoglobin is not an enzyme and is thus not catalyzing a reaction, the similarity of the plots is not coincidental. In both cases, the binding of an external molecule is being measured &ndash directly, in the hemoglobin plot, and indirectly by the V0 vs. [S] plot, since substrate binding is a factor in enzyme reaction velocity.

Allosteric inhibition

Allosterically, regulation of these enzymes works by inducing different physical states (shapes, as it were) that affect their ability to bind to substrate. When an enzyme is inhibited by binding an effector, it is converted to the T-state (T=tight), it has a reduced affinity for substrate and it is through this means that the reaction is slowed.

Allosteric activation

On the other hand, when an enzyme is activated by effector binding, it converts to the R-state (R=relaxed) and binds substrate much more readily. When no effector is present, the enzyme may be in a mixture of T- and R-states.

Feedback inhibition

An interesting kind of allosteric control is exhibited by HMG-CoA reductase, which catalyzes an important reaction in the pathway leading to the synthesis of cholesterol. Binding of cholesterol to the enzyme reduces the enzyme&rsquos activity significantly. Cholesterol is not a substrate for the enzyme, so it is therefore a heterotropic effector.

Notably, though, cholesterol is the end-product of the pathway that HMG-CoA reductase catalyzes a reaction in. When enzymes are inhibited by an end-product of the pathway in which they participate, they are said to exhibit feedback inhibition.

Feedback inhibition always operates by allosterism and further, provides important and efficient control of an entire pathway. By inhibiting an early enzyme in a pathway, the flow of materials (and ATP hydrolysis required for their processing) for the entire pathway is stopped or reduced, assuming there are not alternate supply methods.

Pathway control

In the cholesterol biosynthesis pathway, stopping this one enzyme has the effect of shutting off (or at least slowing down) the entire pathway. This is significant because after catalysis by HMG-CoA reductase, there are over 20 further reactions necessary to make cholesterol, many of them requiring ATP energy. Shutting down one reactions stops all of them. Another excellent example of allosteric control and feedback inhibition is the enzyme ATCase, discussed below.

Another interesting example of allosteric control and feedback inhibition is associated with the enzyme Aspartate Transcarbamoylase (ATCase). This enzyme, which catalyzes a step in the synthesis of pyrimidine nucleotides, has 12 subunits. These include six identical catalytic subunits and six identical regulatory subunits. The catalytic subunits bind to substrate and catalyze a reaction. The regulatory subunits bind to either ATP or CTP. If they bind to ATP, the enzyme subunits arrange themselves in the R-state.

Figure 4.45 - Schematic structure of ATCase. Regulatory Units = R, Catalytic Units = C. Image by Aleia Kim

The R-state of ATCase allows the substrate to have easier access to the six active sites and the reaction occurs more rapidly. For the same amount of substrate, an enzyme in the R-state will have a higher velocity than the same enzyme that is not in the R-state. By contrast, if the enzyme binds to CTP on one of its regulatory subunits, the subunits will arrange in the T-state and in this form, the substrate will not have easy access to the active sites, resulting in a slower velocity for the same concentration of substrate compared to the R-state. ATCase is interesting in that it can also flip into the R-state when one of the substrates (aspartate) binds to an active site within one of the catalytic subunits.

Aspartate has the effect of activating the catalytic action of the enzyme by favoring the R-state. Thus, aspartate, which is a substrate of the enzyme is a homotropic effector and ATP and CTP, which are not substrates of the enzyme are heterotropic effectors of ATCase.

Figure 4.46 - Plots of V0 vs. [S] for ATCase. Left - Allosteric effect of aspartate. Right - Allosteric effects of ATP (activator) and CTP (inhibitor). Image by Pehr Jacobson

Enzyme Kinetics – Michaelis–Menten kinetics

In 1913, Linor Michaelis (1875-1949) and Maud Menten (1879-1960) put forward the enzyme-substrate complex theory. According, the enzyme (E) combines with the substrate(S), to form an enzyme-substrate(ES) complex, which immediately breaks down to the Enzyme and the Product (P).

The above reactions are assumed to be reversible. Here k1, k2, k3, k4 are specific rate constants. Michelis-Menton equation is the rate equation for the reaction catalyzed by an enzyme having a single substrate. In this derivation that the Brigg’s and Halden.

  • Molar Concentration of [E] =Concentration of free (or) free (or) uncombined enzyme
  • [ES]=Concentration of Enzyme-Substrate complex
  • [Et]=Total enzyme concentration (the sum of the free and combined forms)
  • [S]=Concentration of Substrate
  • [P]=Concentration of Product

The substrate concentration is assumed to be far greater than the concentration of Enzyme [E]. So that the amount of substrate-bound by the enzyme at any given time is negligible. Compared with the total concentration of [S].

Systematic studies of the effect of substrate concentration on ionic enzyme activity began performing in the late nineteenth century.

Already in 1882, the concept of an enzyme-substrate complex as an intermediary in the process of enzymatic catalysis was introduced. In 1913, Leonor Michaelis (pictured left) and Maud Menten (pictured right) developed this theory and proposed a rate equation that explains the kinetic behavior of the enzyme.

To explain the observed relation between the initial velocity (v0) and the initial substrate concentration ([S]0) Michaelis and Menten proposed that the enzymatically catalyzed reactions occur in two phases :

  • The first phase: In the first step , the enzyme-substrate complex is formed.
  • Second Phase: The enzyme-substrate complex results in the formation of the product, releasing the free enzyme.

In this scheme, k 1 , k 2 and k 3 are constants each individual kinetics of the process and also are called microscopic rate constants . Accordingly, we can say that:

One can distinguish between free enzyme (E) and attached to the enzyme substrate (S), so that the total concentration of enzyme , [E T ], (which is constant throughout the reaction) is:

As [E] = [ET] – [ES], it follows that:

This kinetic model adopts the steady-state hypothesis , according to which the concentration of the enzyme-substrate complex is small and constant throughout the reaction (Figure, right).

Therefore, the rate of formation of the enzyme-substrate complex (v 1 ) is equal to that of its dissociation (v 2 + v 3 ):

Further, as [ES] is constant, the rate of formation of the products is constant:

As v 1 = v 2 + v 3 , we can say that:

k 1 [S] [E T ] – k 1 [S] [ES] = k 2 [ES] + k 3 [ES]

Solving for [ES], is that: being km=(k2+k3) / k1, where the expression (k 2 + k 3 ) / k 1 has been replaced by K M , or Michaelis-Menten constant .

This link gives us an explanation of the reasons that make the K M an important kinetic parameter .

Thus, at steady state, the rate of formation of the product is:

For any enzyme, [E reaction T], k 3 and KM are constants. Let us consider two extreme cases:

  • A small substrate concentrations ([S] << KM ) = v (k3[ET] /KM) [S]. As the terms in parentheses are constant, they can be included in a new constant, kobs, so that the expression is reduced to v = k obs [S], so that the reaction is a first-order kinetic process.
  • A high substrate concentrations ([S] >> KM), v = k 3 [E T ] . The reaction rate is independent of the concentration of the substrate, and therefore, the reaction is a zero-order kinetic process. In addition, both k 3 and [E T ] are constant and allows us to define a new parameter, the maximum reaction rate (V max ): V max = k 3 [E T ], which is the rate that would be achieved when all available enzyme bound to the substrate is at.

If we introduce the parameter V max in the general rate equation (boxed formula above), we obtain the best known of the Michaelis-Menten expression :

There are enzymes that do not obey the Michaelis-Menten equation. It says it’s not Michaelian kinetics. This happens with Allosteric enzymes, whose graph v vs. [S] is not hyperbole, but a sigmoid (figure right). The Sigmoidal kinetics, small variations in [S] in a critical area (near KM ) results in large variations in the reaction rate.

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Hayley Milliman is a former teacher turned writer who blogs about education, history, and technology. When she was a teacher, Hayley's students regularly scored in the 99th percentile thanks to her passion for making topics digestible and accessible. In addition to her work for PrepScholar, Hayley is the author of Museum Hack's Guide to History's Fiercest Females.

Watch the video: Enzyme activity, specific activity, turn over no, Katal (May 2022).


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