4-Aminobutyrate aminotransferase reaction of sulfhydryl residues connected with catalytic activity.
4-Aminobutyrate aminotransferase is inactivated by preincubation with N-(1-pyrene)maleimide (mixing molar ratio 10:1) at pH 7. The reaction with N-(1-pyrene)maleimide was monitored by fluorescence spectroscopy and the degree of labeling of the enzyme determined by absorption spectroscopy. The blocking of 2 cysteinyl residues/enzyme dimer is needed for inactivation of the aminotransferase. The time course of the reaction is significantly affected by the substrate alpha-ketoglutarate, which afforded complete protection against the loss of catalytic activity. Trypsin digestion of pyrene-labeled aminotransferase, followed by gel filtration and "fingerprint" analysis, revealed the presence of only one peptide tagged with the fluorescent probe. The reaction of approximately 1.9 SH residues/dimer with iodosobenzoate resulted in enzyme inactivation together with a formation of an oligomeric species of Mr = 100,000 detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cross-linked subunits are dissociated by addition of 2-mercaptoethanol which also restores full catalytic activity. Altogether, these observations are consistent with the concept that inactivation of 4-aminobutyrate aminotransferase by iodosobenzoate proceeds through disulfide bond formation between vicinal cysteinyl residues of the protein. It is postulated that the critical sulfhydryl groups of the enzyme are situated on opposite sides of the dimeric structure at the subunit interfaces.
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007463:Iodobenzoates / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D007463:Iodobenzoates; D008301:Maleimides; D013438:Sulfhydryl Compounds; C008154:2-iodosobenzoic acid; C003944:N-(3-pyrene)maleimide; D000612:4-Aminobutyrate Transaminase; D014357:Trypsin