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What are the units of enzyme activity?

What are the units of enzyme activity?


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I was looking at this graph of turnip peroxidase activity and I saw that they use units of 1/sec for enzyme activity. What does this unit intuitively represent and how is it calculated?


This refers to the turnover number (a.k.a kcat or k2) of an enzyme and is usually calculated using Michaelis-Menten kinetics. Jump to the summary at the end if you want a simple answer. If you want a more thorough answer, consider the following chemical equation:

[E] + [S] ⇌ [ES] → [E] + [P]

This says that a certain concentration of enzyme mixed with a certain concentration of substrate will first combine and form a complex depending on the enzyme's affinity for the substrate. Then the enzyme complex will generate a product depending on the enzyme's ability to convert the transition state into product. The k1 constant is the arrow moving from [E] + [S] → [ES] and the k-1 is the arrow moving from [ES] → [E] + [S] (they oppose each other). The k2 constant is the arrow moving from [ES] → [E] + [P].

Vo = [ES]k2

  • Vo - this is the rate that product is being formed, which can be measured.
  • [ES] - this is the concentration of enzyme-substrate complexes.
  • k2 - this is a constant value comparing the two.

This can also be expressed in terms of maximum rate:

Vmax = [E]Tk2

  • Vmax - Vmax is catalytic rate when [E]T = [ES] (when all of the enzyme present is bound to substrate/saturated).
  • [E]T - [E]T is the total enzyme concentration. [E]T = [E] + [ES].
  • k2 - the kcat is the catalytic constant. It is a constant indicating how quickly an enzyme can convert substrates into products. It is easily observed by this equation.

Summary

So k2 is basically an indicator of how efficiently or quickly an enzyme operates. Not all enzymes follow standard Michaelis-Menten kinetics. For example, the allosteric properties of some enzymes cause a non-linear saturation curve. Because of this, the turnover number is commonly referred to. The turnover number's units of s-1 indicate one product molecule per second, so a turnover rate of 3000 means you can create 3000 products in 1 second at Vmax.


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


Lipase acts on triacylglycerols and releases fatty acids. Determination of its activity is calculated by titration of free fatty acids.

Substrate preparation

  • A buffer solution containing Na2HPO4/NaH2PO4 50 mM, pH=7, is prepared.
  • Next an olive oil emulsion (40 ml of olive oil are added to 60 ml of gum arabic – emulsifier solution 5%, w/v) is prepared and the mixture is homogenized in laboratory homogenizer.
  • The substrate of the enzyme is composed of 50 ml of olive oil emulsion in 45 ml of buffer solution.

Determination of enzymatic activity

  • 0.5 ml of crude enzyme solution is added into 9.5 ml of substrate.
  • The mixture is incubated in a shaker for 1 h at T=28 °C
  • This is followed by titration of the mixture with NaOH solution 50 mM until pH=9

A unit of activity of lipase is the quantity of the enzyme that releases 1μmole of fatty acids in 1 h at T=28 °C.


Enzymes

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


Brent Cornell


Various factors may affect the activity of enzymes, by either affecting the frequency of enzyme-substrate collisions or by affecting the capacity for the enzyme and substrate to interact (e.g. denaturation)

  • Temperature, pH and substrate concentration will all influence the rate of activity of an enzyme

Temperature

  • Low temperatures result in insufficient thermal energy for the activation of an enzyme-catalysed reaction to proceed
  • Increasing the temperature will increase the speed and motion of both enzyme and substrate, resulting in higher enzyme activity
  • This is because a higher kinetic energy will result in more frequent collisions between the enzymes and substrates
  • At an optimal temperature (may vary for different enzymes), the rate of enzyme activity will be at its peak
  • Higher temperatures will cause enzyme stability to decrease, as the thermal energy disrupts the enzyme’s hydrogen bonds
  • This causes the enzyme (particularly the active site) to lose its shape, resulting in the loss of activity (denaturation)

The Effect of Temperature on Enzyme Activity

  • Changing the pH will alter the charge of the enzyme, which in turn will alter protein solubility and overall shape
  • Changing the shape or charge of the active site will diminish its ability to bind the substrate, abrogating enzyme function
  • Enzymes have an optimal pH (may differ between enzymes) and moving outside this range diminishes enzyme activity

The Effect of pH on Enzyme Activity


Substrate Concentration

  • Increasing substrate concentration will increase the activity of a corresponding enzyme
  • More substrates mean there is an increased chance of enzyme and substrate colliding and reacting within a given period
  • After a certain point, the rate of activity will cease to rise regardless of any further increases in substrate levels
  • This is because the environment is saturated with substrate and all enzymes are bound and reacting (V max )

The Effect of Substrate Concentration on Enzyme Activity


Enzyme activity

deficient diversional activity a nursing diagnosis approved by the North American Nursing Diagnosis Association, defined as the experiencing by an individual of decreased stimulation from, interest in, or engagement in recreational or leisure activities. Formerly called diversional activity deficit. Possible causes include prolonged hospitalization or immobility at home, frequent and lengthy treatments such as renal dialysis, and a monotonous, nonstimulating environment. The patient usually gives subjective evidence that this condition exists by verbalizing a feeling of boredom or stating a desire for something to do or gives objective evidence by acting depressed or restless.

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The quantity or concentration of an enzyme can be expressed in molar amounts, as with any other chemical, or in terms of activity in enzyme units.

Enzyme activity Edit

Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s −1 , but this is an excessively large unit. A more practical and commonly used value is enzyme unit (U) = 1 μmol min −1 . 1 U corresponds to 16.67 nanokatals. [1]

Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as gelatin, then measured in gelatin digesting units (GDU), or milk proteins, then measured in milk clotting units (MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU. [2]

An increased amount of substrate will increase the rate of reaction with enzymes, however once past a certain point, the rate of reaction will level out because the amount of active sites available has stayed constant.

Specific activity Edit

The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min −1 mg −1 ). Specific activity gives a measurement of enzyme purity in the mixture. It is the micro moles of product formed by an enzyme in a given amount of time (minutes) under given conditions per milligram of total proteins. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal/kg, but a more practical unit is μmol/mgmin.

Specific activity is a measure of enzyme processivity (the capability of enzyme to be processed), at a specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme.

An active site titration process can be done for the elimination of errors arising from differences in cultivation batches and/or misfolded enzyme and similar issues. This is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible inhibitor. The specific activity should then be expressed as μmol min −1 mg −1 active enzyme. If the molecular weight of the enzyme is known, the turnover number, or μmol product per second per μmol of active enzyme, can be calculated from the specific activity. The turnover number can be visualized as the number of times each enzyme molecule carries out its catalytic cycle per second.

Related terminology Edit

The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L −1 s −1 ).

The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated and then getting a clear result.

All enzyme assays measure either the consumption of substrate or production of product over time. [3] A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments: [4]

  • Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. [3] Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation that the amount of free substrate is approximately equal to the amount of the initial substrate can be made. [5][6] The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.
  • Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. Progress curve experiments were widely used in the early period of enzyme kinetics, but are less common now.
  • Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require specialist techniques (such as flash photolysis of caged compounds) or rapid mixing (such as stopped-flow, quenched flow or continuous flow).
  • Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.

Spectrophotometric Edit

In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, and these are called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.

UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidized forms. An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme. [7]

Direct versus coupled assays

Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here, the product of one reaction is used as the substrate of another, easily detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase.

Fluorometric Edit

Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light.

An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase. [8] Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase or 4-methylumbelliferyl-butyrate for assaying Candida rugosa lipase. [9]

Calorimetric Edit

Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assay in any other way. [10]

Chemiluminescent Edit

Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected.

The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.

Light scattering Edit

Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate. Hence the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.

Microscale thermophoresis Edit

Microscale thermophoresis (MST) [11] measures the size, charge and hydration entropy of molecules/substrates at equilibrium. [12] The thermophoretic movement of a fluorescently labeled substrate changes significantly as it is modified by an enzyme. This enzymatic activity can be measured with high time resolution in real time. [13] The material consumption of the all optical MST method is very low, only 5 μl sample volume and 10nM enzyme concentration are needed to measure the enzymatic rate constants for activity and inhibition. MST allows analysts to measure the modification of two different substrates at once (multiplexing) if both substrates are labeled with different fluorophores. Thus substrate competition experiments can be performed.

Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.

Radiometric Edit

Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes most frequently used in these assays are 14 C, 32 P, 35 S and 125 I. Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).

Radioactivity is usually measured in these procedures using a scintillation counter.

Chromatographic Edit

Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher than HPLC instruments (see High-performance liquid chromatography#Pump pressure). [14]

Several factors effect the assay outcome and a recent review summarizes the various parameters that needs to be monitored to keep an assay up and running. [15]


Nomenclature

An enzyme will interact with only one type of substance or group of substances, called the substrate, to catalyze a certain kind of reaction. Because of this specificity, enzymes often have been named by adding the suffix “-ase” to the substrate’s name (as in urease, which catalyzes the breakdown of urea). Not all enzymes have been named in this manner, however, and to ease the confusion surrounding enzyme nomenclature, a classification system has been developed based on the type of reaction the enzyme catalyzes. There are six principal categories and their reactions: (1) oxidoreductases, which are involved in electron transfer (2) transferases, which transfer a chemical group from one substance to another (3) hydrolases, which cleave the substrate by uptake of a water molecule (hydrolysis) (4) lyases, which form double bonds by adding or removing a chemical group (5) isomerases, which transfer a group within a molecule to form an isomer and (6) ligases, or synthetases, which couple the formation of various chemical bonds to the breakdown of a pyrophosphate bond in adenosine triphosphate or a similar nucleotide.


Enzymes Graphics

Energy is a one of the “Big Ideas” of AP Biology and is also included in the Next Generation Science standards. Students don’t usually learn about the laws of thermodynamics until they take chemistry of physics. Most biology books do have a chapter on cellular metabolism, usually near chapters on cellular respiration. Beginning biology students can become overwhelmed if too much emphasis is placed on the chemistry aspects of the equations, but I’ve found that even my freshman can grasp the basic concepts of enzymes, substrates and activation energy of a reaction. A popular enzyme activity in my Intro Bio class involves putting hydrogen peroxide on liver and observing the bubbles, which are the products.

AP Biology classes must go into more detail about reaction rates and how enzymes work at optimal temperatures and pH. For those classes, I use lactase as a focus enzyme as students explore the properties of enzymes. HHMI has excellent resources on human evolution and lactase persistence which I also include in the unit.

This worksheet can be used as a supplement to other enzyme activities, where students examine graphics showing properties of enzymes. First, they label the enzyme, substrate, active site, and products. Then they view a graph showing energy changes with and without an enzyme, revealing how enzymes lower activation energy. Students also examine a graph showing the optimal pH of pepsin and lipase. Finally, a graphic illustrates how competitive inhibition and allosteric inhibition can reduce the speed of enzymatic reactions.


Sources of enzymes

Purchasing enzymes
The National Centre for Biotechnology Education at the University of Reading in the UK supplies a range of different digestive enzymes that can be used for experiments. The enzymes they use are: savinase and alcalase (both proteases), termamyl (amylase), lipolase (lipase), and celluzyme (cellulase).

Natural sources
You can get protease activity from natural products like kiwifruit and pineapple. We suggest that you crush the fruits in a buffer and then strain them to remove the fruit pulp. The filtered solution will contain a range of cellular molecules, including some proteases.

Another natural source of digestive enzymes is the pancreas, which can be collected from an abattoir and blended with buffer.


Watch the video: Units Of Enzyme Activity (October 2022).