Inhibitors

BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 8

Inhibition Of Enzyme Activity

Biological systems are steady-state systems. This means that they have some capabilities of self-regulation. Cellular metabolic schemes often include long chains of enzyme-catalyzed reactions [catenary systems]. If mechanisms exist for regulating a critical reaction, then the entire chain of reactions may be regulated. One example of regulatory mechanisms is that of product inhibition.

Inhibition may be of two kinds, reversible and irreversible. Reversible inhibitory mechanisms usually have high activity when the concentration of the inhibitor is low and low activity when the concentration of the inhibitor is high. Irreversible inhibition usually results from a covalent and permanent modification of a functional group on the enzyme, rendering the molecule inactive.

There are four generally recognized classes of reversible inhibition. In competitive inhibition, the inhibitor competes with the substrate at the active site on the enzyme. Competitive inhibitors often share structural resemblances with the substrate. Noncompetitive inhibition results from the interaction of the inhibitor and the enzyme at some site other than the substrate binding site. The inhibitor may bind to a functional group on the enzyme in such a manner that the active site is contorted so as not to bind the substrate properly. Uncompetitive inhibition results from the combination of the inhibitor with the enzyme-substrate complex directly, without the formation of an enzyme-inhibitor complex. Mixed inhibition may be considered a consequence of several kinds of inhibition.

One can estimate inhibitor constants, Ki, which are quantitative measures of the inhibitory strength of reagents. One determines a Ki by first performing a standard Km analysis. One next repeats the entire procedure with the inhibitor in the assay buffer. At least two concentrations of inhibitor should be tested.

Inhibitors

1. Prepare enough reagents for three Km determinations.
2. Take 50 mL of assay buffer and add inhibitor to the highest concentration [for example, 0.12 mM].
3. Take 20 mL of this and dilute with 20 mL of standard buffer [final: 0.06 mM].
4. Perform three Km determinations using these three concentrations of inhibitor [0.0 mM, 0.06 mM, 0.12 mM].
5. Plot data with Lineweaver-Burke plots.
6. Determine type of inhibition.
7. Calculate Ki's.

References:

Dixon, M., 1953. The determination of enzyme inhibitor constants. Biochem. J. 55: 170 - 171.
Dixon, M., and E. C. Webb, 1953. Enzymes, 2nd Ed., pp. 315 - 359.

Assignment:

Review enzyme inhibition, using the general texts listed under General Enzymology.

Calculation of Ki

The uninhibited run will provide values of Km and Vmax for the reaction. The inhibited runs will provide apparent values of Km and Vmax, ie., Kmapp and Vmaxapp. Ki is calculated for competitive and noncompetitive inhibition using the following equations:
Competitive inhibition: Ki = [Km] [I] / Kmapp - Km
Noncompetitive inhibition: Ki = [Vmaxapp.] [I] / Vmax

Sample Calculation

Competitive inhibition was observed with NADPH versus NADP⁺ in assays using Drosophila 6PGD. Three runs were performed:

0.0 mM NADP⁺,
0.06 mM NADP⁺,
0.12 mM NADP⁺,

with the following results:

[NADPH] Km / Kmapp
0.00 mM 0.0197789
0.06 mM 0.0392886
0.12 mM 0.0529214

Ki = Km X [I] / Kmapp - Km

For 0.06 mM NADPH:

Ki = [0.0197789] [0.06] / 0.0392886 - 0.0197789
= 0.0011867 / 0.0195097
= 0.0608261 mM

For 0.12 mM NADPH:

Ki = 0.0716119 mM

Mean Ki = 0.066219 mM

Ki = 6.62 X 10-5 M

Figure 12. Inhibition of cauliflower isocitrate dehydrogenase by NADPH.

Figure 13. Inhibition of cauliflower isocitrate dehydrogenase by oxaloacetate and glyoxylate.

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