The Initial Velocity of an Enzyme Reaction

The basic experiment in the study of enzyme behavior is measurement of the appearance of product as a function of time (Fig. 11.5). This is often called an enzyme assay. The product appears most rapidly at the very beginning of the reaction. As the reaction progresses, the rate at which the product appears slows down and eventually becomes zero when the system has reached equilibrium. All reactions are in principle reversible; thus the observed overall rate of the reaction is actually the difference between the rate at which the product is being formed and the rate at which the product is being broken down again in the reverse reaction. In many enzyme reactions the equilibrium lies strongly toward the product.

IN DEPTH 11.1 What to Measure in an Enzyme Assay

In an enzyme assay we measure the appearance of product as a function of time. In principle we could do this by starting identical reactions in a series of test tubes and stopping the reaction in each test tube at a different time after the start and then measuring the amount of product in each tube.

However, if either the substrate or the product has a property that can be measured while the reaction is proceeding, then the whole assay can be performed in one test tube. Many enzyme assays are done by using changes in the absorption of light when the substrate is converted to product. Other optical properties can be used. In their original work Michaelis and Menten studied an enzyme that breaks the disaccharide sucrose down into glucose and fructose. The mixture of glucose plus fructose rotated polarized light differently from sucrose, and they used this property to follow the course of the reaction. If there is no convenient optical property, then others may be available; for example, one can monitor the progress of a reaction in which the polysaccharide glycogen (page 30) is hydrolyzed by digestive enzymes by measuring the resulting fall in viscosity.

We can simplify the analysis of enzyme reactions if we consider only the start of the reaction, when there is no product present and so the back reaction can be neglected. The rate of reaction at time zero (the initial velocity v0, sometimes called the initial rate) is found by plotting a graph of product concentration as a function of time and measuring the slope at time zero (Fig. 11.5). In practice the slope is measured over the first 5% of the total reaction. Initial velocity, v0, is conveniently expressed as the rate at which the concentration of the product increases, that is, moles per liter per second.

Enzymes must be assayed under controlled conditions because temperature, pH, and other factors alter the activity. Most are such very effective catalysts that they must be assayed under conditions where the concentration of the enzyme is always very much less than the concentration of the substrate. Otherwise the reaction would be over in a fraction of a second.

Figure 11.5. Definition of v0, the initial velocity of a chemical reaction.

Enzymes are studied for many reasons. An understanding of how they achieve their catalytic excellence and specificity is of fundamental interest and has many practical applications as we increasingly use them in industrial processes and we even seek to design enzymes for particular tasks. Measurements of enzyme concentrations and properties allow us to study processes within cells and organisms. The starting point of any study of an enzyme is to determine its activity, measured as kcat, and how tightly it can bind its substrate—its substrate affinity. Substrate affinity is measured by another constant called the Michaelis constant, KM. We will now explain how these constants are measured.

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