Identification of subsidiary catalytic groups at the active site of beta-ketoacyl-CoA thiolase by covalent modification of the protein.
beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzyme's amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.
D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives; D000101:Acetyl-CoA C-Acetyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000123:Acetyltransferases / Q000378:metabolism*; D000214:Acyl Coenzyme A; D000588:Amines / Q000378:metabolism; D000812:Anhydrides / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine
D000214:Acyl Coenzyme A; D000588:Amines; D000812:Anhydrides; D013438:Sulfhydryl Compounds; C010667:acetoacetyl CoA; C014674:methyl methanethiosulfonate; D000105:Acetyl Coenzyme A; D008741:Methyl Methanesulfonate; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase; D003545:Cysteine