N-15 ISOTOPE EFFECTS ON NONENZYMATIC AND ASPARTATE-TRANSCARBAMYLASE CATALYZED-REACTIONS OF CARBAMYL-PHOSPHATE

Kinetic isotope effects at the amide nitrogen of carbamyl phosphate were measured to determine whether catalysis by aspartate transcarbamylase proceeds through a cyanic acid or tetrahedral intermediate. Decomposition of the mono- and dianion of carbamyl phosphate was used as models for reaction of carbamyl phosphate proceeding through a tetrahedral adduct or cyanic acid intermediate, respectively. The N-15 kinetic isotope effects for decomposition of the mono- (1.0028) and dianion (1.0105) of carbamyl phosphate were sufficiently different to permit a distinction to be made between a cyanic acid intermediate or tetrahedral adduct. The intrinsic N-15 kinetic isotope effects for the aspartate transcarbamylase catalyzed reaction were determined with an active site mutant of aspartate transcarbamylase in which histidine 134 was replaced with alanine (1.0027) and with the wild-type enzyme with cysteine sulfinate as substrate in place of aspartate (1.0024). In both of these systems a full C-13 intrinsic isotope effect has been previously observed (Waldrop et al. Biochemistry, in press; Parmentier et al. Biochemistry, in press). The similarity of these isotope effects to the isotope effect for the decomposition of the carbamyl phosphate monoanion demonstrates that cyanic acid is not an intermediate in the enzyme catalyzed reaction. The observed N-15 kinetic isotope effect for the wild-type enzyme with a low level of aspartate as substrate was 1.0014, a value consistent with the previously measured commitment for carbamyl phosphate (Parmentier et al. Biochemistry, in press). P-31 NMR analysis of phosphate from reactions run in 50% (H2O)-O-18 showed that catalysis by aspartate transcarbamylase involves C-O bond cleavage, thus ruling out carbamic acid as a potential intermediate. We conclude that catalysis by aspartate transcarbamylase proceeds via a tetrahedral adduct.