BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 6. INTRODUCTION TO ENZYMOLOGY

6.14. Competitive and Noncompetitive Inhibition Differ in Kinetics

Measuring catalysis rates at various substrate concentrations makes it possible to distinguish between competitive and noncompetitive inhibition. In competitive inhibition, on a plot of 1/V versus 1/[S], the straight lines intersect the vertical axis at the same point regardless of the presence of the inhibitor; only the slope of the line changes (Fig. 6.17). This demonstrates that V_mах remains unchanged in competitive inhibition. A characteristic feature of competitive inhibition is that it can be overcome by a sufficiently high concentration of substrate. Indeed, the substrate and the inhibitor compete for the same site. At a sufficiently high Substrate Concentration, nearly all active sites are occupied by the substrate, and the enzyme exhibits full activity. The increase in the slope of the line on the 1/V versus 1/[S] plot reflects the binding affinity of the competitive inhibitor. In the presence of a competitive inhibitor, equation (16) is replaced by the following:

Class="center">(24)

where [I] is the inhibitor concentration and K_i is the dissociation constant of the enzyme-inhibitor complex

(25)

In other words, in the presence of a competitive inhibitor, the slope of the line increases by a factor of . Consider an enzyme with a K_m of 10^-4 M. In the absence of an inhibitor, V = V_mах/2 at [S] = 10^-4 M. In the presence of 2 • 10^-3 M of a competitive inhibitor that binds to the enzyme with K_i = 10^-3 M, the apparent K_m becomes 3 • 10^-4 M. Consequently, V = V_mах/4.

Fig. 6.17. Double-reciprocal plot of enzyme kinetics in the presence or absence of a competitive inhibitor. V_mах is unchanged, while K_m increases

In noncompetitive inhibition (Fig. 6.18), V_mах decreases, which means that the line intercepts the vertical axis at a higher point. The slope of the line, equal to K_m/V^I_mах, increases proportionally. Unlike V_max, K_m is unaffected by this type of inhibition. Increasing the substrate concentration does not overcome noncompetitive inhibition. The maximum reaction velocity V^I_max in the presence of a noncompetitive inhibitor is given by the equation

(26)

Fig. 6.18. Double-reciprocal plot of enzyme kinetics in the presence or absence (O—O) of a noncompetitive inhibitor. A noncompetitive inhibitor has no effect on K_m, but decreases V_mах

6.15. Treatment of Ethylene Glycol Poisoning Based on Competitive Inhibition

Each year, about 50 people die from Ethylene glycol poisoning, an additive in automotive antifreeze. Ethylene glycol itself is not lethally toxic; rather, the actual poison is its oxidation product, oxalic acid. The first step in its conversion is The oxidation of ethylene glycol by Alcohol dehydrogenase (Fig. 6.19). This reaction can be effectively inhibited by administering a large, nearly toxic dose of ethanol. The MECHANISM OF ACTION is that ethanol acts as a competitive substrate, thereby blocking the oxidation of ethylene glycol into aldehyde derivatives. The ethylene glycol is then excreted harmlessly. The same principle underlies the Treatment of methanol poisoning.

Fig. 6.19. Ethanol inhibits The formation of oxalic acid from ethylene glycol

6.16. Allosteric Enzymes Do Not Obey Michaelis-Menten Kinetics

The Michaelis-Menten model has had a profound impact on The Development of enzymology. Its primary advantage lies in its simplicity and broad applicability. However, not all Enzymes obey Michaelis-Menten kinetics. Most notably, a large group of allosteric enzymes exhibits a sigmoidal dependence of reaction velocity V on substrate concentration [S], rather than the hyperbolic curve predicted by the Michaelis-Menten Equation [equation (15)]. Recall that the oxygen-binding curve for Myoglobin is hyperbolic, whereas for Hemoglobin it is sigmoidal. The situation with enzymes is entirely analogous. In allosteric enzymes, one Active Site within the enzyme molecule influences another active site in the same molecule. As a result of this subunit interaction, substrate binding becomes cooperative, and the V versus [S] curve adopts a sigmoidal shape. Furthermore, The activity of allosteric Enzymes can be regulated by specific molecules that bind to the enzyme at noncatalytic sites, much like how hemoglobin's oxygen binding is affected by bisphosphoglycerate, H⁺, and CO₂.