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. 1983 Sep 1;213(3):635–642. doi: 10.1042/bj2130635

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate.

P F Leadlay, J Q Fuller
PMCID: PMC1152178  PMID: 6311169

Abstract

(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.

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Selected References

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  1. ALLEN S. H., KELLERMEYER R., STJERNHOLM R., JACOBSON B., WOOD H. G. The isolation, purification, and properties of methylmalonyl racemase. J Biol Chem. 1963 May;238:1637–1642. [PubMed] [Google Scholar]
  2. Albery W. J., Knowles J. R. Deuterium and tritium exchange in enzyme kinetics. Biochemistry. 1976 Dec 14;15(25):5588–5600. doi: 10.1021/bi00670a025. [DOI] [PubMed] [Google Scholar]
  3. Arnstadt K. I., Schindlbeck G., Lynen F. Zum Mechanismus der Kondensationsreaktion der Fettsäurebiosynthese. Eur J Biochem. 1975 Jul 15;55(3):561–571. doi: 10.1111/j.1432-1033.1975.tb02193.x. [DOI] [PubMed] [Google Scholar]
  4. Bodanszky A., Bodanszky M. Sepharose-avidin column for the binding of biotin or biotin-containing peptides. Experientia. 1970 Mar 15;26(3):327–327. doi: 10.1007/BF01900128. [DOI] [PubMed] [Google Scholar]
  5. Cardinale G. J., Abeles R. H. Purification and mechanism of action of proline racemase. Biochemistry. 1968 Nov;7(11):3970–3978. doi: 10.1021/bi00851a026. [DOI] [PubMed] [Google Scholar]
  6. Cheung Y. F., Fung C. H., Walsh C. Stereochemistry of propionyl-coenzyme A and pyruvate carboxylations catalyzed by transcarboxylase. Biochemistry. 1975 Jul;14(13):2981–2986. doi: 10.1021/bi00684a029. [DOI] [PubMed] [Google Scholar]
  7. Cuatrecasas P., Wilchek M., Anfinsen C. B. Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci U S A. 1968 Oct;61(2):636–643. doi: 10.1073/pnas.61.2.636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuller J. Q., Leadlay P. F. Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. The reaction of (2R)-methylmalonyl-CoA in tritiated water. Biochem J. 1983 Sep 1;213(3):643–650. doi: 10.1042/bj2130643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Herlihy J. M., Maister S. G., Albery W. J., Knowles J. R. Energetics of triosephosphate isomerase: the fate of the 1(R)-3H label of tritiated dihydroxyacetone phsophate in the isomerase reaction. Biochemistry. 1976 Dec 14;15(25):5601–5607. doi: 10.1021/bi00670a026. [DOI] [PubMed] [Google Scholar]
  10. Kenyon G. L., Hegeman G. D. Mandelate racemase. Adv Enzymol Relat Areas Mol Biol. 1979;50:325–360. doi: 10.1002/9780470122952.ch7. [DOI] [PubMed] [Google Scholar]
  11. MAZUMDER R., SASAKAWA T., KAZIRO Y., OCHOA S. Metabolism of propionic acid in animal tissues. IX. Methylmalonyl coenzyme A racemase. J Biol Chem. 1962 Oct;237:3065–3068. [PubMed] [Google Scholar]
  12. Northrop D. B. Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry. 1975 Jun 17;14(12):2644–2651. doi: 10.1021/bi00683a013. [DOI] [PubMed] [Google Scholar]
  13. OVERATH P., STADTMAN E. R., KELLERMAN G. M., LYNEN F. [On the mechanism of the transformation of methylmalonyl-CoA into succinyl-CoA. III. Purification and properties of methylmalonyl-CoA-isomerase]. Biochem Z. 1962;336:77–98. [PubMed] [Google Scholar]
  14. Retey J., Lynen F. The absolute configuration of methylmalonyl-CoA. Biochem Biophys Res Commun. 1964 Jul 1;16(4):358–361. doi: 10.1016/0006-291x(64)90040-3. [DOI] [PubMed] [Google Scholar]
  15. Rudnick G., Abeles R. H. Reaction mechanism and structure of the active site of proline racemase. Biochemistry. 1975 Oct 7;14(20):4515–4522. doi: 10.1021/bi00691a028. [DOI] [PubMed] [Google Scholar]
  16. Sprecher M., Clark M. J., Sprinson D. B. The absolute configuration of methylmalonyl coenzyme A and stereochemistry of the methymalonyl coenzyme A mutase reaction. J Biol Chem. 1966 Feb 25;241(4):872–877. [PubMed] [Google Scholar]
  17. Wood H. G., Chiao J. P., Poto E. M. A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits. J Biol Chem. 1977 Feb 25;252(4):1490–1499. [PubMed] [Google Scholar]
  18. YOSHIDA A. PURIFICATION AND CHEMICAL CHARACTERIZATION OF MALATE DEHYDROGENASE OF BACILLUS SUBTILIS. J Biol Chem. 1965 Mar;240:1113–1117. [PubMed] [Google Scholar]

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