Abstract
β-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of β-ketothiolase with a molecular weight of 190000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44000, 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 K m value for acetoacetyl coenzyme A in the thiolysis reaction was 10 μM, and that for coenzyme A was 8.5 μM. The apparent K m 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.
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Abbreviations
- PHB:
-
poly-β-hydroxybutyrate
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Nishimura, T., Saito, T. & Tomita, K. Purification and properties of β-ketothiolase from Zoogloea ramigera . Arch. Microbiol. 116, 21–27 (1978). https://doi.org/10.1007/BF00408729
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DOI: https://doi.org/10.1007/BF00408729