Index to this page
Link to a general discussion of other
aspects of enzyme structure and function.

Enzyme Kinetics

Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process.

They achieve their effect by temporarily binding to the substrate and, in doing so, lowering the activation energy needed to convert it to a product.

The rate at which an enzyme works is influenced by several factors, e.g., The study of the rate at which an enzyme works is called enzyme kinetics. Let us examine enzyme kinetics as a function of the concentration of substrate available to the enzyme. Plotting Vi as a function of [S], we find that

Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).



Plotting the reciprocals of the same data points yields a "double-reciprocal" or Lineweaver-Burk plot. This provides a more precise way to determine Vmax and Km.

The Effects of Enzyme Inhibitors

Enzymes can be inhibited

The distinction can be determined by plotting enzyme activity with and without the inhibitor present.

Competitive Inhibition

In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence. So while Vmax can still be reached if sufficient substrate is available, one-half Vmax requires a higher [S] than before and thus Km is larger.

Noncompetitive Inhibition

With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so This Lineweaver-Burk plot displays these results.

An Example

When a slice of apple is exposed to air, it quickly turns brown. This is because the enzyme o-diphenol oxidase catalyzes the oxidation of phenols in the apple to dark-colored products. (A similar enzyme, tyrosinase, converts tyrosine to melanin.)

Let us determine:

Preparing for the Assay:

First Experiment: No Inhibitor

  Tube A Tube B Tube C Tube D
[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM
1/[S] 0.21 0.83 1.67 3.33
Delta OD540
(Vi)
0.081 0.048 0.035 0.020
1/Vi 12.3 20.8 31.7 50.0

The table above summarizes the results.

Making a Lineweaver-Burk plot of these results shows (red) that

Second Experiment: Effect of para-hydroxybenzoic acid (PHBA)

As before, but this time add a fixed amount of a solution of PHBA to each of the four tubes.

The table below summarizes the results.

  Tube A Tube B Tube C Tube D
[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM
1/[S] 0.21 0.83 1.67 3.33
Delta OD540
(Vi)
0.060 0.032 0.019 0.011
1/Vi 16.7 31.3 52.6 90.9

The Lineweaver-Burk plot of these results is shown above in green.

Third Experiment: Effect of phenylthiourea

As before, but this time add a fixed amount of a solution of phenylthiourea in each of the four tubes.

The table below summarizes the results.

  Tube A Tube B Tube C Tube D
[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM
1/[S] 0.21 0.83 1.67 3.33
Delta OD540
(Vi)
0.040 0.024 0.016 0.010
1/Vi 25 41 62 102

The Lineweaver-Burk plot of these results is shown above in blue.

Summary

Here, then, is a method by which catalytic power of different enzymes can be compared.

The table gives Km values (mM) for several enzymes - some of which you can encounter with links to other pages on this site.

Enzyme Substrate Km (mM)
Catalase H2O2 1,100
Chymotrypsin Gly-Tyr-Gly 108
Carbonic anhydrase CO2 12
beta-galactosidase D-lactose 4
Acetylcholinesterase acetylcholine (ACh) 0.09
beta-lactamase benzylpenicillin 0.02
Link to a general discussion of other aspects of enzyme structure and function.
Welcome&Next Search

12 March 2001