Reversible dissociation of active octamer of cyanase to inactive dimer promoted by alteration of the sulfhydryl group.
Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate resulting in the decomposition of cyanate to ammonia and bicarbonate. In this study, the role of the single sulfhydryl group in each of the eight identical subunits of cyanase was investigated. Tetranitromethane, methyl methanethiosulfonate, N-ethylmaleimide, and Hg2+ all reacted with the sulfhydryl group to give derivatives which had reduced activities and which dissociated reversibly to inactive dimer. Association of inactive dimer to active octamer was facilitated by the presence of azide (cyanate analog) and bicarbonate, increased temperature and enzyme concentration, and presence of phosphate. Nitration of tyrosine residues by tetranitromethane occurred only in the absence of azide and bicarbonate, suggesting that at least some of the tyrosine residues become exposed when octamer dissociates to dimer. Site-directed mutagenesis was used to prepare a mutant enzyme in which serine was substituted for cysteine. The mutant enzyme was catalytically active and had properties very similar to native enzyme, except that it was less stable to treatment with urea and to high temperatures. These results establish that in native cyanase the sulfhydryl group per se is not required for catalytic activity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity.
D000619:Aminohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001386:Azides / Q000494:pharmacology; D001639:Bicarbonates / Q000494:pharmacology; D019759:Carbon-Nitrogen Lyases; D002850:Chromatography, Gel; D003545:Cysteine; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D046911:Macromolecular Substances; D008627:Mercuric Chloride / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D009154:Mutation; D010070:Oxalates / Q000378:metabolism; D019815:Oxalic Acid; D012694:Serine; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds; D013774:Tetranitromethane / Q000494:pharmacology
D001386:Azides; D001639:Bicarbonates; D004793:Enzyme Reactivators; D046911:Macromolecular Substances; D010070:Oxalates; D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; D012694:Serine; D008627:Mercuric Chloride; D019815:Oxalic Acid; D008741:Methyl Methanesulfonate; D000619:Aminohydrolases; C019953:cyanate hydrolase; D019759:Carbon-Nitrogen Lyases; D013774:Tetranitromethane; D003545:Cysteine; D005033:Ethylmaleimide