NOVEL USES OF ISOTOPE EFFECTS TO ELUCIDATE ENZYME REACTION MECHANISMS

A new technique (Multiple Isotope Effects) for determining enzyme reaction mechanisms and intrinsic isotope effects has been developed. By determining the ('13)C isotope effect on V/K with both a deuterated and unlabeled substrate, and the deuterium isotope effect on V/K, it is possible to tell whether the ('13)C-sensitive and deuterium-sensitive steps are the same or not and, if they are different, to determine which comes first in the mechanism. If the two isotope-sensitive steps are the same, (1) deuteration increases the size of the observed ('13)C isotope effect, and (2) the intrinsic ('13)C and deuterium isotope effects, and the commitments can be calculated. When the deuterium- and ('13)C-sensitive steps are different, deuteration decreases the size of the observed ('13)C isotope effect. The data fit the equation {('13)(V/K)(,H) - 1}/{('13)(V/K)(,D) - 1} = ('D)(V/K)/('D)K(,eq) when the deuterium-sensitive step comes first, but fit the equation {('13)(V/K)(,H) - ('13)K(,eq)}/{('13)(V/K)(,D) - ('13)K(,eq)} = ('D)(V/K) when the ('13)C-sensitive step comes first.;Application of the Multiple Isotope Effect technique to glucose-6-phosphate dehydrogenase, using its ('13)C and deuterium isotope effects at C-1, allowed calculation of the intrinsic isotope effects ('D)k = 5.27, ('13)k = 1.0408, and ('(alpha)-D)k = 1.054, and commitments c(,f) = 0.75 and c(,r) = 0.49.;The malic enzyme reaction illustrates the ability of this technique to distinguish concerted from stepwise reaction mechanisms. For this enzyme both concerted and stepwise (through an oxalacetate intermediate) reactions can be formulated. It was found that ('13)(V/K)(,H) was greater than ('13)(V/K)(,D), verifying a stepwise mechanism in which hydride transfer precedes decarboxylation.;The NAD-dependent oxidative decarboxylation of prephenate catalyzed by chorismate mutase-prephenate dehydrogenase was also studied. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI.;With formate dehydrogenase the intrinsic deuterium, (alpha)-secondary deuterium, primary ('13)C (at formate), and ('18)O isotope effects were measured as a function of changing nucleotide substrate. As the nucleotide becomes more redox-positive and the overall reaction more spontaneous, the transition state becomes earlier (more reactant-like). The (alpha)-secondary deuterium effects (substitution at C-4 of the nucleotide) were determined as a function of nucleotide, and a correlation is seen between V(,max) and the magnitude of this effect. It was also observed that primary deuterium substitution decreased the size of this (alpha)-secondary isotope effect for all the nucleotides investigated. These results are interpreted in terms of quantum mechanical tunneling.