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.