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.