title: Proton nuclear magnetic resonance investigation of the mechanism of flavin C-4a adduct formation induced by oxidized nicotinamide adenine dinucleotide binding to monoalkylated pig heart lipoamide dehydrogenase.
abstract: The active center thiol of monoalkylated pig heart lipoamide dehydrogenase, EHR, is induced to form an adduct to the enzyme-bound flavin adenine dinucleotide (FAD) at the C-4a position upon binding oxidized nicotinamide adenine dinucleotide (NAD+) [Thorpe, C., & Williams, C. H., Jr. (1976) J. Biol. Chem. 251, 7726-7728]. In light of hypotheses on covalent electron transfer between pyridine nucleotide and flavin, the induction of the thiol-flavin C-4a adduct by NAD+ is reasonably envisioned as involving a covalent bond between the modified flavin and the NAD+. The double-resonance proton nuclear magnetic resonance technique of cross saturation was used to probe the existence of covalent bond formation between the modified flavin of EHR and its inducer molecule, NAD+. Cross-saturation of the free NAD+ signals was not observed even though the spin-lattice relaxation time of NAD+ and the rate of exchange between free NAD+ and NAD+ bound to EHR were well within the limits required for cross-saturation. We conclude that a noncovalent interaction between NAD+ and FAD induces the formation of the thiol-flavin C-4a covalent adduct in EHR. A model by which NAD+ binding induces nucleophilic attack by the nascent thiolate of EHR is discussed.
mesh_terms: D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013552:Swine
chemical_list: D009243:NAD; D008058:Dihydrolipoamide Dehydrogenase