@article {5082,
	title = {Active site residues of glutamate racemase},
	journal = {Biochemistry},
	volume = {40},
	number = {21},
	year = {2001},
	note = {ISI Document Delivery No.: 436GCTimes Cited: 44Cited Reference Count: 38},
	month = {May},
	pages = {6199-6204},
	type = {Article},
	abstract = {Glutamate racemase, MurI, catalyzes the interconversion of glutamate enantiomers in a cofactor-independent fashion and provides bacteria with a source of D-Glu for use in peptidoglycan biosynthesis. The enzyme uses a "two-base" mechanism involving a deprotonation of the substrate at the ct-position to form an anionic intermediate, followed by a reprotonation in the opposite stereochemical sense. In the Lactobacillus fermenti enzyme, Cys73 is responsible for the deprotonation of D-glutamate, and Cys184 is responsible for the deprotonation of L-glutamate; however, very little is known about the roles of other active site residues. This work describes the preparation of four mutants in which strictly conserved residues containing ionizable side chains were modified (D10N, D36N, E152Q, and H186N). During the course of this research, the structural analysis of a crystallized glutamate racemase indicated that three of these residues (D10, E152, and H186) are in the active site of the enzyme [Hwang, K. Y., Cho, C.-S., Kim, S. S., Sung, H.-C., Yu, Y. G., and Cho, Y. (1999) Nat. Struct. Biol. 6, 422-426]. Two of the mutants, D10N and H186N, displayed a marked decrease in the values of k(cat), but not K-M, and are therefore implicated as important catalytic residues. Further analysis of the primary kinetic isotope effects observed with a-deuterated substrates showed that a significant asymmetry was introduced into the free energy profile by these two mutations. This is interpreted as evidence that the mutated residues normally assist the catalytic thiols in acting as bases (D10 with C73 and H186 with C184). An alternate possibility is that the residues may serve to stabilize the carbanionic intermediate in the racemization reaction.},
	keywords = {AMINO-ACID RACEMASE, AQUIFEX PYROPHILUS, CARBON ACIDS, ENZYME-CATALYZED REACTIONS, ESCHERICHIA-COLI, INFLUENZAE DIAMINOPIMELATE EPIMERASE, IRREVERSIBLE INHIBITOR, MANDELATE RACEMASE, PROLINE, PROTON ABSTRACTION, RACEMASE},
	isbn = {0006-2960},
	url = {<Go to ISI>://000168927300002},
	author = {Glavas, S. and Tanner, M. E.}
}
@article {4557,
	title = {Catalytic acid/base residues of glutamate racemase},
	journal = {Biochemistry},
	volume = {38},
	number = {13},
	year = {1999},
	note = {ISI Document Delivery No.: 182QZTimes Cited: 43Cited Reference Count: 37},
	month = {Mar},
	pages = {4106-4113},
	type = {Article},
	abstract = {Glutamate racemase is a cofactor-independent enzyme that employs two active-site cysteine residues as acid/base catalysts during the interconversion of glutamate enantiomers, In a given reaction direction, a thiolate from one of the cysteines abstracts the alpha-proton, and the other cysteine thiol delivers a proton to the opposite face of the resulting carbanionic intermediate. This paper reports that the C73S and C184S mutants are still capable of racemizing glutamate with specificity constants about 10(3)-fold lower than those of the wild-type enzyme, A "one-base requiring" reaction, the elimination of water from N-hydroxyglutamate, has been used to deduce which thiol acts as the base for a given enantiomer. With D-N-hydroxyglutamate the C73S mutant is a much poorer catalyst than wild-type enzyme, whereas the C184S mutant is a somewhat better catalyst. This trend was reversed with L-N-hydroxyglutamate, suggesting that Cys73 is responsible for the deprotonation of D-glutamate and Cys184 is responsible for the deprotonation of L-glutamate. In addition, with C73S the V-max/K-M isotope effect on D-glutamate racemization was greater than that seen with wild-type enzyme, whereas the isotope effect with L-glutamate had decreased. The results were reversed with the C184S mutant. This is interpreted as being due to an asymmetry in the free energy profiles that is induced upon mutation, with the deprotonation step involving a serine becoming the more cleanly rate-determining of the two. These results support the above assignment and the notion that a carbanionic intermediate is formed during catalysis.},
	keywords = {BACILLUS-STEAROTHERMOPHILUS, DIAMINOPIMELATE EPIMERASE, ENERGETICS, ESCHERICHIA-COLI, INHIBITOR, IRREVERSIBLE, L-ALANINE, MANDELATE RACEMASE, MECHANISM, PROLINE RACEMASE, PURIFICATION},
	isbn = {0006-2960},
	url = {<Go to ISI>://000079510800031},
	author = {Glavas, S. and Tanner, M. E.}
}
