Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide.
D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for function. The purified enzyme devoid of lipid (apodehydrogenase) is inactive but can be reactivated by forming a complex with phospholipid containing lecithin. We find that, of the six half cysteines present in D-beta-hydroxybutyrate dehydrogenase, only two are in the reduced form and available for modification with N-ethylmaleimide, even after denaturation in sodium dodecyl sulfate. Diamide treatment of either the inactive apodehydrogenase or the active enzyme-phospholipid complex resulted in complete loss of enzymic activity, the apodehydrogenase being assayed after addition of phospholipid. The inactivation by diamide can be reversed by the addition of dithiothreitol with full recovery of activity. Derivatization using N-[14C]ethylmaleimide showed that diamide modified only one sulfhydryl per enzyme monomer. The other sulfhydryl appears not to be essential for function since full activity can be restored after this sulfhydryl had been covalently derivatized with N-ethylmaleimide. Protein cross-linking was not observed after diamide modification of D-beta-hydroxybutyrate dehydrogenase, indicating that a disulfide bridge was not formed between enzyme subunits. The diamide-modified enzyme retains the ability to bind coenzyme, NAD(H), as detected by quenching of the intrinsic fluorescence of the protein. However, resonance energy transfer from protein to bound NADH and enhancement of NADH fluorescence were not observed, indicating that diamide modification of the protein alters the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
D001391:Azo Compounds / Q000494:pharmacology*; D003958:Diamide / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D013997:Time Factors
D001391:Azo Compounds; D009243:NAD; D003958:Diamide; D006884:Hydroxybutyrate Dehydrogenase; D005033:Ethylmaleimide; D004229:Dithiothreitol