Abstract
We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specific lipoamide dehydrogenase produced during growth on valine (molecular weight, 49,000). Branched-chain keto acid dehydrogenase required L-valine, oxidized nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, and magnesium chloride. A partially purified preparation catalyzed the oxidation of 2-keto-[1-14C]isovalerate to [14C]carbon dioxide, isobutyryl-coenzyme A, and reduced nicotinamide adenine dinucleotide in equimolar amounts. Both the Km and the Vmax for 2-ketoisovalerate were affected by the addition of L-valine to the assay mixture. However, only the Vmax values for oxidized nicotinamide adenine dinucleotide and coenzyme A were affected when L-valine was present. This suggested that valine acted by affecting the binding of branched-chain keto acids to subunit E1 of the complex.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Connelly J. L., Danner D. J., Bowden J. A. Branched chain alpha-keto acid metabolism. I. Isolation, purification, and partial characterization of bovine liver alpha-ketoisocaproic:alpha-keto-beta-methylvaleric acid dehydrogenase. J Biol Chem. 1968 Mar 25;243(6):1198–1203. [PubMed] [Google Scholar]
- DANCIS J., HUTZLER J., LEVITZ M. THE DIAGNOSIS OF MAPLE SYRUP URINE DISEASE (BRANCHED- CHAIN KETOACIDURIA) BY THE IN VITRO STUDY OF THE PERIPHERAL LEUKOCYTE. Pediatrics. 1963 Aug;32:234–238. [PubMed] [Google Scholar]
- Danner D. J., Lemmon S. K., Besharse J. C., Elsas L. J., 2nd Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria. J Biol Chem. 1979 Jun 25;254(12):5522–5526. [PubMed] [Google Scholar]
- Martin R. R., Marshall V. D., Sokatch J. R., Unger L. Common enzymes of branched-chain amino acid catabolism in Pseudomonas putida. J Bacteriol. 1973 Jul;115(1):198–204. doi: 10.1128/jb.115.1.198-204.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Massey L. K., Sokatch J. R., Conrad R. S. Branched-chain amino acid catabolism in bacteria. Bacteriol Rev. 1976 Mar;40(1):42–54. doi: 10.1128/br.40.1.42-54.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Namba Y., Yoshizawa K., Ejima A., Hayashi T., Kaneda T. Coenzyme A- and nicotinamide adenine dinucleotide-dependent branched chain alpha-keto acid dehydrogenase. I. Purification and properties of the enzyme from Bacillus subtilis. J Biol Chem. 1969 Aug 25;244(16):4437–4447. [PubMed] [Google Scholar]
- Parker P. J., Randle P. J. Partial purification and properties of branched-chain 2-oxo acid dehydrogenase of ox liver. Biochem J. 1978 Jun 1;171(3):751–757. doi: 10.1042/bj1710751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pettit F. H., Yeaman S. J., Reed L. J. Purification and characterization of branched chain alpha-keto acid dehydrogenase complex of bovine kidney. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4881–4885. doi: 10.1073/pnas.75.10.4881. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roberts C. M., Sokatch J. R. Branched chain amino acids as activators of branched chain ketoacid dehydrogenase. Biochem Biophys Res Commun. 1978 Jun 14;82(3):828–833. doi: 10.1016/0006-291x(78)90857-4. [DOI] [PubMed] [Google Scholar]
- Rüdiger H. W., Langenbeck U., Goedde H. W. Oxidation of branched chain -ketoacids in Streptococcus faecalis and it's dependence on lipoic acid. Hoppe Seylers Z Physiol Chem. 1972 Jun;353(6):875–882. doi: 10.1515/bchm2.1972.353.1.875. [DOI] [PubMed] [Google Scholar]
- Sokatch J. R., McCully V., Gebrosky J., Sokatch D. J. Isolation of a specific lipoamide dehydrogenase for a branched-chain keto acid dehydrogenase from Pseudomonas putida. J Bacteriol. 1981 Nov;148(2):639–646. doi: 10.1128/jb.148.2.639-646.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]