Use of isotope effects to determine enzymatic and nonenzymatic mechanisms

Kinetic and equilibrium isotope effects were determined to study the chemical mechanism and/or transition state structures of several reactions. The lanthanide ion catalyzed cyclization of uridine-3'- p-nitrophenyl phosphate was studied by 15N isotope effects at the nitro group. The transition state of these cyclization reactions has very little bond order to the leaving group. Equilibrium heavy atom isotope effects between several molecules were determined and placed within a growing framework of known equilibrium effects. These equilibrium effects aided in the interpretation of kinetic 13C and 15N isotope effects with aspartate aminotransferase. The multiple isotope effect method was used with this enzyme to study the nature of the proton shift between the internal aldimine and the ketimine. The results were inconclusive, but the steps contributing to the observed isotope effects were identified. Heavy atom isotope effects with the tyrosine-225 to phenylalanine mutant showed that tyrosine-225 plays an important role in ketimine hydrolysis. Kinetic 13C and 15N isotope effects were used to study the nonenzymatic decarboxylation of picolinic acid and N-methyl picolinic acid, and the mechanism of orotidylate decarboxylase. The results indicate that orotidylate decarboxylase does not employ a zwitterion intermediate. Kinetic 15N isotope effects with D-amino acid oxidase suggest that the mechanism of this enzyme does not involve attack of a carbanion on FAD. Kinetic 15N isotope effects with carbamyl phosphate synthetase indicate that breakdown of the tetrahedral intermediate is rate-limiting. The isotope effects are reduced with the glutamate-841 to lysine mutant, suggesting that this amino acid plays a role in a regulatory conformational change prior to ammonia formation.