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In effect, a competitive inhibitor acts by decreasing the number of free enzyme molecules available to bind substrate, ie, to form ES, and thus eventually to form product, as described below:

A competitive inhibitor and substrate exert reciprocal effects on the concentration of the EI and ES complexes. Since binding substrate removes free enzyme available to combine with inhibitor, increasing the [S] decreases the concentration of the EI complex and raises the reaction velocity. The extent to which [S] must be increased to completely overcome the inhibition depends upon the concentration of inhibitor present, its affinity for the enzyme K, and the Km of the enzyme for its substrate.

Double Reciprocal Plots Facilitate the Evaluation of Inhibitors

Double reciprocal plots distinguish between competitive and noncompetitive inhibitors and simplify evaluation of inhibition constants Ki. vi is determined at several substrate concentrations both in the presence and in the absence of inhibitor. For classic competitive inhibition, the lines that connect the experimental data points meet at the y axis (Figure 8-9). Since the y intercept is equal to 1/Vmax, this pattern indicates that when 1/[S] approaches 0, v is independent of the presence of inhibitor. Note, however, that the intercept on the x axis does vary with inhibitor concentration—and that since -1/Km' is smaller than 1/Km, Km' (the "apparent Km") becomes larger in the presence of increasing concentrations of inhibitor. Thus, a competitive inhibitor has no effect on Vm^ but raises K'm, the apparent Km for the substrate.

Figure 8-9. Lineweaver-Burk plot of competitive inhibition. Note the complete relief of inhibition at high [S] (ie, low 1/[S]).

For simple competitive inhibition, the intercept on the x axis is

Once Km has been determined in the absence of inhibitor, K can be calculated from equation (47). K values are used to compare different inhibitors of the same enzyme. The lower the value for Ki, the more effective the inhibitor. For example, the statin drugs that act as competitive inhibitors of HMG-CoA reductase (Chapter 26) have K values several orders of magnitude lower than the Km for the substrate HMG-CoA.

Simple Noncompetitive Inhibitors Lower Vmax but Do Not Affect Km

In noncompetitive inhibition, binding of the inhibitor does not affect binding of substrate. Formation of both EI and EIS complexes is therefore possible. However, while the enzyme-inhibitor complex can still bind substrate, its efficiency at transforming substrate to product, reflected by Vmax, is decreased. Noncompetitive inhibitors bind enzymes at sites distinct from the substrate-binding site and generally bear little or no structural resemblance to the substrate.

For simple noncompetitive inhibition, E and EI possess identical affinity for substrate, and the EIS complex generates product at a negligible rate (Figure 8-10). More complex noncompetitive inhibition occurs when binding of the inhibitor does affect the apparent affinity of the enzyme for substrate, causing the lines to intercept in either the third or fourth quadrants of a double reciprocal plot (not shown).

J_

v,

X/

__1_

V max i ^ Km

Figure 8-10. Lineweaver-Burk plot for simple non-competitive inhibition.

Irreversible Inhibitors "Poison" Enzymes

In the above examples, the inhibitors form a dissociable, dynamic complex with the enzyme. Fully active enzyme can therefore be recovered simply by removing the inhibitor from the surrounding medium. However, a variety of other inhibitors act irreversibly by chemically modifying the enzyme. These modifications generally involve making or breaking covalent bonds with aminoacyl residues essential for substrate binding, catalysis, or maintenance of the enzyme's functional conformation. Since these covalent changes are relatively stable, an enzyme that has been "poisoned" by an irreversible inhibitor remains inhibited even after removal of the remaining inhibitor from the surrounding medium.

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Diabetes 2

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