Overview

Enzymes speed up reactions by lowering the activation energy of the reactants. The speed at which the enzyme turns reactants into products is called the rate of reaction. Several factors impact the rate of reaction, including the number of available reactants. Enzyme kinetics is the study of how an enzyme changes the rate of a reaction.

Scientists typically study enzyme kinetics with a fixed amount of enzyme in the controlled environment of a test tube. When more reactant, or substrate, is added to a fixed amount of enzyme, the rate of the reaction increases as the enzyme can make more product. As a result, when the reaction rate is graphed against substrate concentration, increasing the substrate increases the reaction rate. However, once all the active sites of the enzyme are occupied, the reaction rate plateaus. The concentration of substrate at which the maximum rate of reaction is reached is called V_max. The number of present enzyme molecules limits V_max. If the amount of enzyme is increased, V_max increases, but adding more substrate has no effect.

The graph of rate of reaction versus substrate concentration can reveal other important characteristics of an enzyme’s kinetics. The substrate concentration at which the reaction rate is halfway to V_max (i.e., ½ V_max) is called the Michaelis constant (K_m). K_m is a representation of the affinity between an enzyme and a substrate. Enzymes with a lower K_m require less substrate to reach V_max and therefore have a higher affinity for their substrate. Interestingly, for many enzymes, the value of K_m is very close to the cellular concentration of the substrate. Near K_m, slight changes in substrate concentration can significantly impact the reaction rate, so small changes in cellular substrate availability can impact the function of an entire biological pathway.

Not all enzymes produce the hyperbolic-shaped substrate-rate graph known as Michaelis Menten kinetics. Michaelis Menten kinetics assumes that the enzyme catalyzes a single substrate. Enzymes that are regulated allosterically have multiple active sites and tend to produce a sigmoid-shaped graph when the reaction rate is plotted versus the substrate concentration.

Procedure

Enzyme kinetics studies the rates of enzyme-catalyzed reactions. The rates of repeated experiments at varying substrate concentrations are monitored by measuring the concentration of substrate consumed or product formed over time.

These results can be graphed to show how substrate concentration affects the rate or velocity of a reaction.

The reaction velocity increases linearly with increasing substrate amounts at low concentrations, but it begins to plateau at higher concentrations. The rate approaches a maximum velocity or V_max – the rate where the enzyme is completely saturated with the substrate.

Enzyme affinity measures how strongly or weakly an enzyme binds its substrate and is quantified by K_M, the Michaelis constant. The value of K_M is equal to the substrate concentration when the rate is 50% of the V_max.

A  small K_M  indicates an enzyme has a high substrate affinity and vice versa. An enzyme with a larger K_M requires higher substrate concentrations to approach its maximum velocity compared to an enzyme with a lower K_M.