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Abstract

Glutamate racemase from Lactobacillus fermenti (murl, E.C. 5.1.1.3) is a cofactorindependent enzyme which catalyzes the interconversion of the enantiomers of glutamate using Cys73 and Cysl84 as the general acid/base catalysts. A cysteine thiolate abstracts the α-proton from one face of glutamate and the other cysteine thiol delivers a proton to the opposite face. The roles of the cysteine residues are explored using the two glutamate racemase mutant enzymes, C73S and C184S. The mutants retain the ability to racemize glutamate with specificity constants ~10³-fold lower than those of wild type enzyme. The mutant-catalyzed dehydration reaction of iV-hydroxyglutamate, a one-base requiring reaction, is used to determine which cysteine residue acts on each enantiomer of glutamate. With [sub D]-N-hydroxyglutamate, the C73S mutant is a poorer catalyst than the wild type enzyme, whereas theC184S mutant is a better catalyst. The opposite trend is observed with L-A^-hydroxyglutamate. The results suggest Cys73 is the residue responsible for the deprotonation of D-glutamate and Cysl84 is responsible for the deprotonation of L-glutamate. Furthermore, the V[sub max]/K[sub m] isotope effect for the C73S mutant in the D —> L reaction direction is larger than that observed for wild type enzyme and smaller in the L —> D reaction direction. The opposite trend is observed for the C184S mutant. Presumably, an asymmetry in the reaction profile is induced by the mutation making the deprotonation step involving the serine residue more cleanly rate determining. This result supports the assigned roles for each of the cysteine residues of glutamate racemase. Further experiments explore the importance of the four strictly conserved residues, AsplO, Asp36, Glul52 and Hisl86, by preparing appropriate mutant enzymes. The effect of each of the D36N and E152Q mutations is to reduce k[sub cat] by a modest factor of ~2- to 3-fold and suggests these residues are not important to catalysis. They do appear to be involved in binding since there is an increase in the K[sub m] value for each of these mutants. The k[sub cat] values for the D10N and H186N mutants, however, are decreased by three orders of magnitude relative to wild type enzyme implying an important catalytic role for these residues. The V[sub max] isotope effects for D10N and H186N are affected in each reaction direction with the ratio between the isotope effects increased to 2.20 ± 0.18 for D10N relative to the wild type ratio of 1.40 ± 0.34, and decreased to 0.70 ± 0.05 for H186N. A possible role for AsplO and Hisl86 is to stabilize the thiolate form of Cys73 and Cysl84, respectively.