Me. Tanner et al., ISOTOPE EFFECTS AND THE IDENTIFICATION OF CATALYTIC RESIDUES IN THE REACTION CATALYZED BY GLUTAMATE RACEMASE, Biochemistry, 32(15), 1993, pp. 3998-4006
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.