Mechanism of transfer of reduced nicotinamide adenine dinucleotide among dehydrogenases. Transfer rates and equilibria with enzyme-enzyme complexes.
The direct transfer of NADH between A-B pairs of dehydrogenases and also the dissociation of NADH from individual E-NADH complexes have been investigated by transient stopped-flow kinetic techniques. Such A-B transfers of NADH occur without the intermediate dissociation of coenzyme into the aqueous solvent environment [Srivastava, D.K., & Bernhard, S.A. (1985) Biochemistry 24, 623-628]. The equilibrium distributions of limiting NADH among aqueous solvent and A and B dehydrogenase sites have also been determined. At sufficiently high but realizable concentrations of dehydrogenases, both the transfer rate and the equilibrium distribution of bound NADH are virtually independent of the excessive enzyme concentrations; at excessive E2 concentration, substantial NADH is bound to the E1 site. These results further substantiate earlier kinetic arguments for the preferential formation of an EA-NADH-EB complex, within which coenzyme is directly transferred between sites. The unimolecular specific rates of coenzyme transfer from site to site are nearly invariant among different A-B dehydrogenase pairs. The equilibrium constants for the distribution of coenzyme within the EA X EB complexes are near unity. At high [E2] and for [E2] greater than [E1] greater than [NADH], E1-NADH X E2 and E1 X NADH-E2 are virtually the only coenzyme-contained species. In contrast to the nearly invariant unimolecular NADH transfer rates within EA X EB complexes, unimolecular specific rates of dissociation of NADH from E-NADH into aqueous solution are highly variable.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008968:Molecular Conformation; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility
D001051:Apoenzymes; D009243:NAD; D005993:Glycerolphosphate Dehydrogenase; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009247:NADH, NADPH Oxidoreductases