ISOTOPE EFFECTS AND THE IDENTIFICATION OF CATALYTIC RESIDUES IN THE REACTION CATALYZED BY GLUTAMATE RACEMASE

Primary kinetic isotope effects on V(max) were observed in both reacti on directions upon racemizing samples of [2-H-2] glutamate with the co factor-independent glutamate racemase from Lactobacillus. This support s a deprotonation/protonation mechanism for racemization in which the breaking of the carbon-hydrogen bond at C-2 is partially rate-determin ing. Substantial ''overshoots'' were observed when the time course of racemization of either enantiomer of glutamate was monitored using cir cular dichroism spectroscopy. This is consistent with a ''two-base'' m echanism accompanied by a kinetic isotope effect. ''Competitive deuter ium washout'' experiments were used to measure kinetic isotope effects on V(max)/K(m) of 2.5 for (S)-glutamate and 3.4 for (R)-glutamate. Th e ratio of the notably different isotope effects was confirmed by ''do uble competitive deuterium washout'' experiments. Site-directed mutage nesis was used to generate the mutant C73A and C184A enzymes. In each case the mutant enzymes were inactive as racemases. The two mutant enz ymes are, however, capable of catalyzing the elimination of HCl from o pposite enantiomers of threo-3-chloroglutamic acid, a process that pre sumably requires only one enzymic base. This finding indicates that th e active sites of the mutant enzymes are intact and that the two cyste ines flank the bound substrate molecule. It appears that cysteine-73 i s responsible for the abstraction of the C-2 hydrogen from (R)-glutama te and cysteine-184 abstracts the proton from (S)-glutamate in the rac emization reaction of the wild-type enzyme.