Advertisement

Archives of Microbiology

, Volume 116, Issue 1, pp 21–27 | Cite as

Purification and properties of β-ketothiolase from Zoogloea ramigera

  • Takeko Nishimura
  • Terumi Saito
  • Kenkichi Tomita
Article

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.

Key words

β-Ketothiolase Zoogloea ramigera I-16-M Thiolysis Ping pong mechanism 

Non-Standard Abbreviation

PHB

poly-β-hydroxybutyrate

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Andrews, P.: Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J. 91, 222–233 (1964)Google Scholar
  2. Beers, R. F., Jr., Sizer, I. W.: A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140 (1952)Google Scholar
  3. Bergmeyer, H. U., Gawehn, K., Klotzsch, H., Krebs, H. A., Williamson, D. H.: Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Biochem. J. 102, 423–431 (1967)Google Scholar
  4. Berndt, H., Schlegel, H. G.: Kinetics and properties of β-ketothiolase from Clostridium pasteurianum. Arch. Microbiol. 103, 21–30 (1975)Google Scholar
  5. Cleland, W. W.: Enzyme kinetics. In: Annu. Rev. Biochem., Vol. 36 (P. D. Boyer, A. Meister, R. L. Sinsheimer, E. E. Snell, eds.), pp. 77–112. Palo Alto: Annual Reviews 1967Google Scholar
  6. Cleland, W. W.: Steady state kinetics. In: The enzymes, Vol. 2 (P. D. Boyer, ed.), pp. 1–65. New York-London: Academic Press 1970Google Scholar
  7. Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., Lane, M. D.: Molecular and catalytic properties of cytosolic acetoacetyl coenzyme A thiolase from avian liver. J. Biol. Chem. 248, 2275–2284 (1973)Google Scholar
  8. Crabtree, K., McCoy, E., Boyle, W. C., Rohlich, G. A.: Isolation, identification, and metabolic role of the sudanophylic granules of Zoogloea ramigera. Appl. Microbiol. 13, 218–226 (1965)Google Scholar
  9. Davis, B. J.: Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404–427 (1964)Google Scholar
  10. Flavin, M.: Methylmalonyl coenzyme A. In: Methods in enzymology, Vol. 6 (S. P. Colowick, N. O. Kaplan, eds.), pp. 538–539. New York-London: Academic Press 1963Google Scholar
  11. Fukui, T., Yoshimoto, A., Matsumoto, M., Hosokawa, S., Saito, T., Nishikawa, H., Tomita, K.: Enzymatic synthesis of poly-β-hydroxybutyrate in Zoogloea ramigera. Arch. Microbiol. 110, 149–156 (1976)Google Scholar
  12. Gehring, U., Riepertinger, C.: Dissoziation und Rekonstitution der Thiolase. European J. Biochem. 6, 281–292 (1968)Google Scholar
  13. Gehring, U., Riepertinger, C., Lynen, F.: Reinigung und Kristallisation der Thiolase, Untersuchungen zum Wirkungsmechanismus. European J. Biochem. 6, 264–280 (1968)Google Scholar
  14. Huth, W., Dierich, C., v. Oeynhausen, V., Seubert, W.: Multiple mitochondrial forms of acetoacetyl-CoA thiolase in rat liver: possible regulatory role in ketogenesis. Biochem. Biophys. Res. Commun. 56, 1069–1077 (1974)Google Scholar
  15. Kornblatt, J. A., rudney, H.: Two forms of acetoacetyl coenzyme A thiolase in yeast. I. Separation and properties. J. Biol. Chem. 246, 4417–4423 (1971)Google Scholar
  16. Layne, E.: Spectrophotometric and turbidimetric methods for measuring proteins. In: Methods in enzymology, Vol. 3 (S. P. Colowick, N. O. Kaplan, eds.), pp. 447–454. New York-London: Academic Press 1957Google Scholar
  17. Martin, R. G., Ames, B. N.: A method for determining the sedimentation behavior of enzyme: Application to protein mixture. J. Biol. Chem. 236, 1372–1379 (1961)Google Scholar
  18. Mazzei, Y., Negrel, R., Ailhaud, G.: Purification and some properties of thiolase from Escherichia coli. Biochim. Biophys. Acta 220, 129–131 (1970)Google Scholar
  19. Middleton, B.: The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver. Biochem. J. 139, 109–121 (1974)Google Scholar
  20. Oeding, V., Schlegel, H. G.: β-Ketothiolase from Hydrogenomonas eutropha H 16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism. Biochem. J. 134, 239–248 (1973)Google Scholar
  21. Racker, E.: Alcohol dehydrogenase from baker's yeast. In Methods in enzymology, Vol. 1 (S. P. Colowick, N. O. Kaplan, eds.), pp. 500–503, New York-London: Academic Press 1955Google Scholar
  22. Ritchie, G. A. F., Senior, P. J., Dawes, E. A.: The purification and characterization of acetoacetyl-coenzyme A reductase from Azotobacter beijerinckii. Biochem. J. 121, 309–316 (1971)Google Scholar
  23. Saito, T., Fukui, T., Ikeda, F., Tanaka, Y., Tomita, K.: An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera. Arch. Microbiol. 114, 211–217 (1977)Google Scholar
  24. Senior, P. J., Dawes, E. A.: The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem. J. 134, 225–238 (1973)Google Scholar
  25. Simon, E., Shemin, D.: The preparation of S-succinyl coenzyme A. J. Am. Chem. Soc. 75, 2520 (1953)Google Scholar
  26. Srere, P. A., Kosicki, G. W.: The purification of citrate-condensing enzyme. J. Biol. Chem. 236, 2557–2559 (1961)Google Scholar
  27. Stern, J. R.: Crystalline crotonase from ox liver. In: Methods in enzymology, Vol. 1 (S. P. Colowick, N. O. Kaplan, eds.), pp. 559–566. New York-London: Academic Press 1955aGoogle Scholar
  28. Stern, J. R.: Enzymes of acetoacetate formation and breakdown. In: Methods in enzymology, Vol. 1 (S. P. Colowick, N. O. Kaplan, eds.), pp. 573–585. New York-London: Academic Press 1955bGoogle Scholar
  29. Tubbs, P. K., Garland, P. B.: Assay of coenzyme A and some acyl derivatives. In: Methods in enzymology, Vol. 13 (J. M. Lowenstein, ed.), pp. 535–551, New York-London: Academic Press 1969Google Scholar
  30. Weber, K., Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412 (1969)Google Scholar

Copyright information

© Springer-Verlag 1978

Authors and Affiliations

  • Takeko Nishimura
    • 1
  • Terumi Saito
    • 1
  • Kenkichi Tomita
    • 1
  1. 1.Department of Health Chemistry, Faculty of Pharmaceutical SciencesKyoto UniversityKyotoJapan

Personalised recommendations