MEDIUM-CHAIN ACYL COA DEHYDROGENASE - EVIDENCE FOR PHOSPHORYLATION

Mature medium chain acyl-CoA dehydrogenase isolated from pig kidney (p kMCADH) and originating from mitochondria carries a phosphate group as demonstrated by P-31-NMR-spectroscopy and chemical analysis. Two broa d resonances at -6.3 and -8 ppm are observed and are assigned to the p yrophosphate 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. Wit h recombinant, human wild type MCADH expressed in and purified from E. coli only the two FAD phosphates (2 +/- 0.35) are found. Similarly, p kMCADH which has been converted to the apoenzyme and reconstituted to holoenzyme also contains 2 +/- 0.4 phosphates. The covalently bound ph osphate can be hydrolyzed by phosphatase and subsequently removed by d ialysis, The phosphate group has no detectable effect on the catalytic activity of the MCADH measured with artificial and natural electron a ccepters such as pig electron transferring flavoprotein. However, phos phorylation has a marked effect on protein solubility which is +5-fold lower for the dephosphorylated protein.