PRODUCT DEPENDENCE OF DEUTERIUM-ISOTOPE EFFECTS IN ENZYME-CATALYZED REACTIONS

Theory for enzyme-catalyzed reactions is developed for the dependence on product concentration of deuterium isotope effects on V and V/K. Generally, a product that decreases the off-rate for a second product to zero causes the isotope effect on V/K to decrease to (D)K(eq) and that on V to decrease to a value between 1 and (D)K(eq). If the second product off-rate is decreased to a finite value, (D)V and D(V/K) will decrease to a value greater than (D)K(eq), while if there is no effect on the off-rate for the second product, (D)V and D(V/K) will not change. Interestingly, for a ping-pong mechanism, the presence of the product that provides a reversible connection between the isotope-sensitive step and the isotope-insensitive half-reaction will give an isotope effect on V/K for the latter. (In the absence of the product, the isotope effect on V/K for the isotope-insensitive half-reaction will be unity.) Theory is supported with data for alcohol and lactate dehydrogenases. For lactate dehydrogenase, D(V/K(pyruvate)) decreases from 1.93 +/- 0.02 at zero to 1.16 +/-0.02 at infinite lactate concentration, while (D)V decreases from a value of 1.75 +/- 0.03 at zero to a value of 0.93 +/- 0.05 at infinite lactate concentration. Thus, it appears that the pathway in which lactate is released first is greatly preferred, but the pathway in which NAD+ is released before lactate is observed at high lactate concentration. For alcohol dehydrogenase, D(V/K(acetaldhyde)) decreases from a value of 2.8 +/- 0.1 at zero to 1.8 +/- 0.1 at infinite concentration of ethanol, while (D)V remains unchanged and equal to unity. The value of unity observed for (D)V is suggestive of a Theorell-Chance mechanism, but the finite value of D(V/K) obtained at high ethanol concentrations indicates the presence of the pathway in which NAD+ is released prior to ethanol. Data for alcohol dehydrogenase are consistent with antagonism of binding between NAD+ and ethanol, that is ethanol and NAD+ increase the off-rates for NAD+ and ethanol from the E:NAD:ethanol ternary complex.