3.13

Introduction to Enzyme Kinetics

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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.

Overview

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Enzyme kinetics studies the rates of biochemical reactions. Scientists monitor the reaction rates for a particular enzymatic reaction at various substrate concentrations. Additional trials with inhibitors or other molecules that affect the reaction rate may also be performed.

The experimenter can then plot the initial reaction rate or velocity (V_o) of a given trial against the substrate concentration ([S]) to obtain a graph of the reaction properties. For many enzymatic reactions involving a single substrate, this data fits the Michaelis-Menten equation, an equation derived by Leonor Michaelis and Maud Menten.

Static equilibrium diagram with force vectors ΣFx=0, ΣFy=0; educational physics concept.

The equation estimates the maximum velocity (V_max) and the Michaelis constant (K_M) for the enzyme being studied and is based on the following assumptions:

No product is present at the start of the reaction.
The rate of enzyme-substrate complex formation equals the rate of dissociation and breakdown into products.
The enzyme concentration is minimal compared to the substrate concentration.
Only the initial reaction rates are measured.
The enzyme is present either in the free form or in the enzyme-substrate complex.

Different rearrangements of the Michaelis-Menten equation, such as the Lineweaver-Burke, Eadie-Hofsteot, and Hanes-Woolf plots, are alternate ways to graph kinetic parameters. The Lineweaver-Burke or double reciprocal plot is often used to estimate the K_Mand the V_max. The plot uses the reciprocals values of the x and y-axis from the Michaelis-Menten plot. Mathematically, the y-intercept equals 1/V_max, and the x-intercept equals −1/K_M.

The Lineweaver-Burke plot can be used to visually differentiate between inhibitor types – competitive, non-competitive, and uncompetitive. Different rearrangements of the Michaelis-Menten equation, such as the Eadie-Hofstee and Hanes-Woolf plots, are also used to determine kinetic parameters.

Transcript

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Enzyme Kinetics

Michaelis Menten Equation

Lineweaver Burke Plot

Enzyme Substrate Complex

Initial Reaction Rate

Substrate Concentration

Maximum Velocity

Michaelis Constant

Competitive Inhibition

Non Competitive Inhibition