Evaluation of cysteine 283 and glutamic acid 284 in the coenzyme binding site of Salmonella typhimurium glutamate dehydrogenase by site-directed mutagenesis and reaction with the nucleotide analogue 2-[4-bromo-2,3-dioxobutyl)thio)-1,N6-ethenoadenosine 2',5'-bisphosphate.
NADP(+)-specific glutamate dehydrogenase of Salmonella typhimurium was previously shown to react irreversibly at the coenzyme site with the nucleotide analogue 2-((4-bromo-2,3-dioxobutyl)thio)-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A 2',5'-DP) yielding a partially active enzyme, and inactivation was attributed to modification of the peptide Leu282-Cys-Glu-Ile-Lys286 (Bansal, A., Dayton, M.A., Zalkin, H., and Colman, R.F. (1989) J. Biol. Chem. 264, 9827-9835). Three mutant enzymes have now been engineered, expressed in Escherichia coli, and purified: the single mutants C283I and E284Q and the double mutant C283I:E284Q. The wild-type and mutant enzymes have similar specific activities and Km values for alpha-ketoglutarate, ammonium ion, and NADPH, indicating that neither cysteine 283 nor glutamic acid 284 is essential for activity. The mutant enzyme E284Q, like wild-type glutamate dehydrogenase, is substantially inactivated by 2-BDB-T epsilon A 2',5'-DP. In contrast, the two cysteine mutant enzymes, C283I and C283I:E284Q, are not inactivated by 2-BDB-T epsilon A 2',5'-DP. Modified tryptic peptides with the sequence Leu-X-Glu(Gln)-Ile-Lys were isolated from wild-type or E284Q enzymes inactivated by 2-BDB-T epsilon A 2',5'-DP. This peptide was absent from digests of active wild-type enzyme modified in the presence of the protectant NADPH and from digests of active C283I enzyme after incubation with 2-BDB-T epsilon A 2',5'-DP. Although it is not required for catalytic activity, cysteine 283 is implicated by the results of the affinity labeling experiments as the reaction target of the nucleotide analogue and is located in the region of the coenzyme binding site.
Haeffner-Gormley
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Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Chen
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ZD
Zalkin
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Colman
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RF
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005969:Glutamate Dehydrogenase / Q000235:genetics*; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012486:Salmonella typhimurium / Q000201:enzymology*
D000345:Affinity Labels; D005971:Glutamates; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; C047570:2-((4-bromo-2,3-dioxobutyl)thio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate; D005969:Glutamate Dehydrogenase; D003545:Cysteine