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. 1975 Dec;124(3):1462–1474. doi: 10.1128/jb.124.3.1462-1474.1975

Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii.

K K Tung, W A Wood
PMCID: PMC236061  PMID: 369

Abstract

Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to beta-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or beta-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in the absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ALLEN S. H., KELLERMEYER R. W., STJERNHOLM R. L., WOOD H. G. PURIFICATION AND PROPERTIES OF ENZYMES INVOLVED IN THE PROPIONIC ACID FERMENTATION. J Bacteriol. 1964 Jan;87:171–187. doi: 10.1128/jb.87.1.171-187.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BALDWIN R. L., WOOD W. A., EMERY R. S. LACTATE METABOLISM BY PEPTOSTREPTOCOCCUS ELSDENII: EVIDENCE FOR LACTYL COENZYME A DEHYDRASE. Biochim Biophys Acta. 1965 Feb 15;97:202–213. doi: 10.1016/0304-4165(65)90084-x. [DOI] [PubMed] [Google Scholar]
  3. Chrambach A., Reisfeld R. A., Wyckoff M., Zaccari J. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal Biochem. 1967 Jul;20(1):150–154. doi: 10.1016/0003-2697(67)90272-2. [DOI] [PubMed] [Google Scholar]
  4. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  5. DEN H., ROBINSON W. G., COON M. J. Enzymatic conversion of beta-hydroxypropionate to malonic semialdehyde. J Biol Chem. 1959 Jul;234(7):1666–1671. [PubMed] [Google Scholar]
  6. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
  7. Garces E., Cleland W. W. Kinetic studies of yeast nucleoside diphosphate kinase. Biochemistry. 1969 Feb;8(2):633–640. doi: 10.1021/bi00830a026. [DOI] [PubMed] [Google Scholar]
  8. Hammerstedt R. H., Möhler H., Decker K. A., Wood W. A. Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase. I. Physical evidence for a three-subunit molecule. J Biol Chem. 1971 Apr 10;246(7):2069–2074. [PubMed] [Google Scholar]
  9. Hedrick J. L., Smith A. J. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys. 1968 Jul;126(1):155–164. doi: 10.1016/0003-9861(68)90569-9. [DOI] [PubMed] [Google Scholar]
  10. Hersh L. B., Jencks W. P. Isolation of an enzyme-coenzyme A intermediate from succinyl coenzyme A-acetoacetate coenzyme A transferase. J Biol Chem. 1967 Jan 25;242(2):339–340. [PubMed] [Google Scholar]
  11. Jakoby W. B. A technique for the crystallization of proteins. Anal Biochem. 1968 Nov;26(2):295–298. doi: 10.1016/0003-2697(68)90340-0. [DOI] [PubMed] [Google Scholar]
  12. LADD J. N., WALKER D. J. The fermentation of lactate and acrylate by the rumen micro-organism LC. Biochem J. 1959 Feb;71(2):364–373. doi: 10.1042/bj0710364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LEWIS D., ELSDEN S. R. The fermentation of L-threonine, L-serine, L-cysteine and acrylic acid by a gram-negative coccus. Biochem J. 1955 Aug;60(4):683–692. doi: 10.1042/bj0600683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. STADTMAN E. R. The coenzyme A transphorase system in Clostridium kluyveri. J Biol Chem. 1953 Jul;203(1):501–512. [PubMed] [Google Scholar]
  15. STADTMAN E. R. The coenzyme A transphorase system in Clostridium kluyveri. J Biol Chem. 1953 Jul;203(1):501–512. [PubMed] [Google Scholar]
  16. STERN J. R., COON M. J., DEL CAMPILLO A., SCHNEIDER M. C. Enzymes of fatty acid metabolism. IV. Preparation and properties of coenzyme A transferase. J Biol Chem. 1956 Jul;221(1):15–31. [PubMed] [Google Scholar]
  17. STERN J. R., DEL CAMPILLO A. Enzymes of fatty acid metabolism. II. Properties of crystalline crotonase. J Biol Chem. 1956 Feb;218(2):985–1002. [PubMed] [Google Scholar]
  18. Sokatch J. R., Sanders L. E., Marshall V. P. Oxidation of methylmalonate semialdehyde to propionyl coenzyme A in Pseudomonas aeruginosa grown on valine. J Biol Chem. 1968 May 25;243(10):2500–2506. [PubMed] [Google Scholar]
  19. Solomon F., Jencks W. P. Identification of an enzyme-gamma-glutamyl coenzyme A intermediate from coenzyme A transferase. J Biol Chem. 1969 Feb 10;244(3):1079–1081. [PubMed] [Google Scholar]
  20. VAGELOS P. R., EARL J. M. Propionic acid metabolism. III. beta-Hydroxypropionyl coenzyme A and malonyl semialdehyde coenzyme A, intermediates in propionate oxidation by Clostridium kluyveri. J Biol Chem. 1959 Sep;234:2272–2280. [PubMed] [Google Scholar]
  21. VAGELOS P. R. Propionic acid metabolism. IV. Synthesis of malonyl coenzyme A. J Biol Chem. 1960 Feb;235:346–350. [PubMed] [Google Scholar]
  22. WILLIAMS D. E., REISFELD R. A. DISC ELECTROPHORESIS IN POLYACRYLAMIDE GELS: EXTENSION TO NEW CONDITIONS OF PH AND BUFFER. Ann N Y Acad Sci. 1964 Dec 28;121:373–381. doi: 10.1111/j.1749-6632.1964.tb14210.x. [DOI] [PubMed] [Google Scholar]
  23. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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