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. 1981 Nov;148(2):639–646. doi: 10.1128/jb.148.2.639-646.1981

Isolation of a specific lipoamide dehydrogenase for a branched-chain keto acid dehydrogenase from Pseudomonas putida.

J R Sokatch, V McCully, J Gebrosky, D J Sokatch
PMCID: PMC216250  PMID: 6895373

Abstract

We purified lipoamide dehydrogenase from cells of Pseudomonas putida PpG2 grown on glucose (LPD-glu) and lipoamide dehydrogenase from cells grown on valine (LPD-val), which contained branched-chain keto acid dehydrogenase. LPD-glu had a molecular weight of 56,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and LPD-val had a molecular weight of 49,000. The pH optimum for LPD-glu for reduced nicotinamide adenine dinucleotide oxidation was 7.4, compared with pH 6.5 for LPD-val. When oxidized nicotinamide adenine dinucleotide was included in the assay mixture, the pH optima were 7.1 and 5.7, respectively. There was also a difference in pH optima between the two enzymes for oxidized nicotinamide adenine dinucleotide reduction, but the Michaelis constants and maximum velocities were similar. A purified preparation of branched-chain keto acid dehydrogenase, which was deficient in lipoamide dehydrogenase, was stimulated 10-fold by LPD-val but not by LPD-glu, which suggested that the branched-chain keto acid dehydrogenase of P. putida has a specific requirement for LPD-val. In contrast, a partially purified preparation of 2-ketoglutarate dehydrogenase that was deficient in lipoamide dehydrogenase was stimulated by LPD-glu but not by LPD-val, indicating that this complex has a specific requirement of LPD-glu.

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Selected References

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

  1. ATKINSON D. E., HATHAWAY J. A., SMITH E. C. KINETICS OF REGULATORY ENZYMES. KINETIC ORDER OF THE YEAST DIPHOSPHOPYRIDINE NUCLEOTIDE ISOCITRATE DEHYDROGENASE REACTION AND A MODEL FOR THE REACTION. J Biol Chem. 1965 Jun;240:2682–2690. [PubMed] [Google Scholar]
  2. Alwine J. C., Russell R. M., Murray K. N. Characterization of an Escherichia coli mutant deficient in dihydrolipoyl dehydrogenase activity. J Bacteriol. 1973 Jul;115(1):1–8. doi: 10.1128/jb.115.1.1-8.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrera C. R., Namihira G., Hamilton L., Munk P., Eley M. H., Linn T. C., Reed L. J. -Keto acid dehydrogenase complexes. XVI. Studies on the subunit structure of the pyruvate dehydrogenase complexes from bovine kidney and heart. Arch Biochem Biophys. 1972 Feb;148(2):343–358. doi: 10.1016/0003-9861(72)90152-x. [DOI] [PubMed] [Google Scholar]
  4. Guest J. R. Aspects of the molecular biology of lipoamide dehydrogenase. Adv Neurol. 1978;21:219–244. [PubMed] [Google Scholar]
  5. Guest J. R., Creaghan I. T. Gene-protein relationships of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and characterization of lipoamide dehydrogenase mutants. J Gen Microbiol. 1973 Mar;75(1):197–210. doi: 10.1099/00221287-75-1-197. [DOI] [PubMed] [Google Scholar]
  6. Guest J. R. Gene-protein relationships of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: Chromosomal location of the lipoamide dehydrogenase gene. J Gen Microbiol. 1974 Feb;80(2):523–532. doi: 10.1099/00221287-80-2-523. [DOI] [PubMed] [Google Scholar]
  7. Jacobson L. A., Bartholomaus R. C., Gunsalus I. C. Repression of malic enzyme by acetate in Pseudomonas. Biochem Biophys Res Commun. 1966 Sep 22;24(6):955–960. doi: 10.1016/0006-291x(66)90343-3. [DOI] [PubMed] [Google Scholar]
  8. LUSTY C. J. LIPOYL DEHYDROGENASE FROM BEEF LIVER MITOCHONDRIA. J Biol Chem. 1963 Oct;238:3443–3452. [PubMed] [Google Scholar]
  9. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  10. MASSEY V., GIBSON Q. H., VEEGER C. Intermediates in the catalytic action of lipoyl dehydrogenase (diaphorase). Biochem J. 1960 Nov;77:341–351. doi: 10.1042/bj0770341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. MASSEY V. The identity of diaphorase and lipoyl dehydrogenase. Biochim Biophys Acta. 1960 Jan 15;37:314–322. doi: 10.1016/0006-3002(60)90239-0. [DOI] [PubMed] [Google Scholar]
  12. MASSEY V., VEEGER C. Studies on the reaction mechanism of lipoyl dehydrogenase. Biochim Biophys Acta. 1961 Mar 18;48:33–47. doi: 10.1016/0006-3002(61)90512-1. [DOI] [PubMed] [Google Scholar]
  13. Marshall V. D., Sokatch J. R. Regulation of valine catabolism in Pseudomonas putida. J Bacteriol. 1972 Jun;110(3):1073–1081. doi: 10.1128/jb.110.3.1073-1081.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pettit F. H., Reed L. J. Alpha-keto acid dehydrogenase complexes. 8. Comparison of dihydrolipoyl dehydrogenases from pyruvate and alpha-ketoglutarate dehydrogenase complexes of Escherichia coli. Proc Natl Acad Sci U S A. 1967 Sep;58(3):1126–1130. doi: 10.1073/pnas.58.3.1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. REED L. J., KOIKE M., LEVITCH M. E., LEACH F. R. Studies on the nature and reactions of protein-bound lipoic acid. J Biol Chem. 1958 May;232(1):143–158. [PubMed] [Google Scholar]
  17. Sakurai Y., Fekuyoshi Y., Hamada M., Hayakawa T., Koike M. Mammalian alpha-keto acid dehydrogenase complexes. VI. Nature of the multiple forms of pig heart lipoamide dehydrogenase. J Biol Chem. 1970 Sep 10;245(17):4453–4462. [PubMed] [Google Scholar]
  18. Scouten W. H., McManus I. R. Microbial lipoamide dehydrogenase. Purification and some characteristics of the enzyme derived from selected microorganisms. Biochim Biophys Acta. 1971 Feb 10;227(2):248–263. doi: 10.1016/0005-2744(71)90058-1. [DOI] [PubMed] [Google Scholar]
  19. Sokatch J. R., McCully V., Roberts C. M. Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida. J Bacteriol. 1981 Nov;148(2):647–652. doi: 10.1128/jb.148.2.647-652.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Visser J., Strating M. Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography. Biochim Biophys Acta. 1975 Mar 28;384(1):69–80. doi: 10.1016/0005-2744(75)90096-0. [DOI] [PubMed] [Google Scholar]
  21. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  22. Wilson J. E. A comparative study of the multiple forms of pig heart lipoyl dehydrogenase. Arch Biochem Biophys. 1971 May;144(1):216–223. doi: 10.1016/0003-9861(71)90471-1. [DOI] [PubMed] [Google Scholar]
  23. v Muiswinkel-Voetberg H., Veeger C. Conformational studies on lipoamide dehydrogenase from pig heart. 4. The binding of NAD + to non-equivalent sites. Eur J Biochem. 1973 Mar 1;33(2):285–291. doi: 10.1111/j.1432-1033.1973.tb02682.x. [DOI] [PubMed] [Google Scholar]

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