Purification and properties of beta-ketothiolase from Zoogloea ramigera.
beta-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of beta-ketothiolase with a molecular weight of 190 000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44 000, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme thus appeared to be a tetramer with identical subunits. The enzyme showed a pH optimum of 7.5 in the condensation reaction, and 8.5 in the thiolysis reaction. The enzyme employed a Bi Bi ping pong mechanism for the forward thiolysis reaction. The apparent Km value for acetoacetyl coenzyme A in the thiolysis reaction was 10 micron, and that for coenzyme A was 8.5 micron. The apparent Km value for acetyl coenzyme A in the condensation reaction was 0.33 mM. The condensation reaction was inhibited by coenzyme A concentrations lower than 0.1 mM. The enzyme was stable in the presence of dithiothreitol and other SH-compounds, but was strongly inhibited by 0.4 mM p-chloromercuribenzoate.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000102:Acetyl-CoA C-Acyltransferase / Q000302:isolation & purification / Q000378:metabolism; D000217:Acyltransferases / Q000302:isolation & purification; D003065:Coenzyme A / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D015045:Zoogloea / Q000201:enzymology*
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D000105:Acetyl Coenzyme A; D000217:Acyltransferases; D000102:Acetyl-CoA C-Acyltransferase; D003065:Coenzyme A