title: Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. beta-Cys325 is a nonessential active site residue.
abstract: Chemical modification experiments have shown that sulfhydryl groups play an important role in the mechanism of action of Escherichia coli succinyl-CoA synthetase. One of these sulfhydryl groups has been localized in the beta-subunit of the enzyme using the coenzyme A affinity analog, CoA disulfide-S,S-dioxide (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4943). Recently, it has been shown that the reactive sulfhydryl group resides in Cys325 (Nishimura, J. S., Mitchell, T., Ybarra, J., and Matula, J. M., submitted to Eur. J. Biochem. for publication). In the present study, we have changed Cys325 to a glycine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is 83% as active as wild type enzyme. In contrast to wild type succinyl-CoA synthetase, the mutant is refractory to chemical modification by CoA disulfide-S,S-dioxide and methyl methanethiolsulfonate. It is also less reactive with N-ethylmaleimide. Thus, beta-Cys325 is a nonessential active site residue.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
authors.forename: C J
authors.initials: CJ
authors.lastname: Mann
authors.forename: S C
authors.initials: SC
authors.lastname: Hardies
authors.forename: J S
authors.initials: JS
authors.lastname: Nishimura
mesh_terms: D001665:Binding Sites; D003066:Coenzyme A Ligases / Q000235:genetics*; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007163:Immunosorbent Techniques; D013389:Succinate-CoA Ligases / Q000235:genetics*
chemical_list: D003066:Coenzyme A Ligases; D013389:Succinate-CoA Ligases; D003545:Cysteine