title: Medium-chain acyl CoA dehydrogenase: evidence for phosphorylation.
abstract: Mature medium chain acyl-CoA dehydrogenase isolated from pig kidney (pkMCADH) and originating from mitochondria carries a phosphate group as demonstrated by 31P-NMR-spectroscopy and chemical analysis. Two broad resonances at -6.3 and -8 ppm are observed and are assigned to the pyrophosphate group of the cofactor FAD. A third, narrow resonance at 4.65 ppm indicates the presence of a phosphomonoester residue. Chemical analysis of intact pkMCADH shows the presence of 3 +/- 0.3 phosphates, those of FAD and of an additional covalently attached phosphate. With recombinant, human wild type MCADH expressed in and purified from E. coli only the two FAD phosphates (2 +/- 0.35) are found. Similarly, pkMCADH which has been converted to the apoenzyme and reconstituted to holoenzyme also contains 2 +/- 0.4 phosphates. The covalently bound phosphate can be hydrolyzed by phosphatase and subsequently removed by dialysis. The phosphate group has no detectable effect on the catalytic activity of the MCADH measured with artificial and natural electron acceptors such as pig electron transferring flavoprotein. However, phosphorylation has a marked effect on protein solubility which is +5-fold lower for the dephosphorylated protein.
authors.affiliation: Fakultät für Biologie, Universität Konstanz, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Macheroux
authors.forename: C
authors.initials: C
authors.lastname: Sanner
authors.forename: H
authors.initials: H
authors.lastname: Büttner
authors.forename: V
authors.initials: V
authors.lastname: Kieweg
authors.forename: H
authors.initials: H
authors.lastname: Rüterjans
authors.forename: S
authors.initials: S
authors.lastname: Ghisla
mesh_terms: D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001059:Apoproteins; D002384:Catalysis; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D010758:Phosphorus / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D012995:Solubility; D012996:Solutions; D013552:Swine; D014783:Viscosity
chemical_list: D001059:Apoproteins; D010761:Phosphorus Radioisotopes; D012996:Solutions; D010758:Phosphorus; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase