Active site residues of glutamate racemase.
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 alpha-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 alpha-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.
Glavas
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Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
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D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003903:Deuterium / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment
D001426:Bacterial Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D001216:Asparagine; D003903:Deuterium; D000591:Amino Acid Isomerases; C062292:glutamate racemase