Probing the role of cysteine residues in the CheR methyltransferase.
The CheR methyltransferase catalyzes the transfer of methyl groups from S-adenosylmethionine to specific glutamyl residues in bacterial chemoreceptor proteins. Studies with sulfhydryl reagents such as p-chloromercuribenzoate, N-ethylmaleimide, and 5,5'-dithiobis(2-nitrobenzoate) suggest that a cysteine residue is required for enzyme activity. The nucleotide sequence of the cheR gene predicts a 288-amino acid protein with cysteine residues at positions 31 and 229. To ascertain the role of these cysteine residues in the structure and function of the enzyme, oligonucleotide-directed mutagenesis was used to change each cysteine to serine. Whereas the Cys229-Ser mutation had essentially no effect on transferase activity, the Cys31-Ser mutation caused an 80% decrease in enzyme activity. The double mutant in which both cysteines were replaced by serines also had markedly reduced transferase activity. Preincubation of the wild type or Cys229-Ser proteins with either S-adenosylmethionine or beta-mercaptoethanol protected it from inhibition by sulfhydryl reagents, whereas prior incubation with the second substrate, the Tar receptor, gave partial protection. From these studies, Cys31 appears to be necessary for enzyme activity, and it seems to be located in the vicinity of the active site.
City College, Department of Chemistry, New York, New York 10031.
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Charles
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D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry* / Q000502:physiology; D004247:DNA; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013696:Temperature
D013438:Sulfhydryl Compounds; D004247:DNA; D004228:Dithionitrobenzoic Acid; D008780:Methyltransferases; C038706:chemotaxis methyltransferase; D003545:Cysteine; D005033:Ethylmaleimide