Complex formation between nucleotides and D-beta-hydroxybutyrate dehydrogenase studied by fluorescence and EPR spectroscopy.
D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-requiring enzyme which specifically requires phosphosphatidylcholine for enzymic activity. The phosphatidylcholine modifies the binding and orientation of the coenzyme, NAD(H), with respect to the enzyme. In the present study, two derivatives of NAD, spin-labeled either at N-6 or C-8 of the adenine ring, were found to be active as coenzyme. The binding affinity of NADH to the enzyme was opitimized by increasing the salt concentration and increasing the pH from 6 to 8, with the pK at 6.8. Monomethylmalonate, a substrate analogue, was found to enhance NADH binding (Kd is reduced from 4 to 1 microM). Sulfite strongly enhances the binding of NAD+ via the enzyme-catalyzed formation of an adduct of sulfite with the nucleotide; the Kd for binding of NAD-sulfite is in the micromolar range, whereas NAD+ binding is more than a magnitude weaker. The binding of spin-labeled NAD(H) was further characterized by EPR spectroscopy. Increased sensitivity and resolution were obtained with the use of NAD(H) analogues perdeuterated in the spin-label moiety. For these analogues bound to D-beta-hydroxybutyrate dehydrogenase in phospholipid vesicles, EPR studies showed the spin-label moiety to be constrained and revealed two distinct components. Increasing the viscosity of the medium by addition of glycerol affected the EPR spectral characteristics of only the component with the smaller resolved averaged hyperfine splitting. The stage is now set to study motional characteristics of the enzyme, using these spin-labeled probes which mimic the coenzyme.
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004735:Energy Transfer; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008764:Methylmalonic Acid / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013447:Sulfites / Q000494:pharmacology
D008081:Liposomes; D010713:Phosphatidylcholines; D013113:Spin Labels; D013447:Sulfites; D009243:NAD; D008764:Methylmalonic Acid; D006884:Hydroxybutyrate Dehydrogenase; D005990:Glycerol