Mechanism of hydride transfer during the reduction of 3-acetylpyridine adenine dinucleotide by NADH catalyzed by the pyridine nucleotide transhydrogenase of Escherichia coli

The pyridine nucleotide transhydrogenase is a proton pump which catalyzes the reversible transfer of a hydride ion equivalent between NAD(+) and NADP(+) coupled to translocation of protons across the cytoplasmic membrane. The enzyme also catalyzes the reduction of the NAD(+) analog 3-acetylpyridine adenine dinucleotide (AcPyAD(+)) by NADH. It has been proposed (Hutton et al. (1994) fur. J. Biochem. 219, 1041-1051) that this reaction requires NADP(H) as an intermediate. Thus, NADP(+) bound at the NADP(H)-binding site on the transhydrogenase would be reduced by NADH and reoxidized by AcPyAD(+) binding alternately to the NAD(H)-binding site. The reduction of AcPyAD(+) by NADPH would be a partial reaction in the reduction of AcPyAD(+) by NADH. Using cytoplasmic membrane vesicles from mutants having elevated activities for transhydrogenation of AcPyAD(+) by NADH in the absence of added NADP(H), the kinetics of reduction of AcPyAD(+) by NADH and NADPH have been compared, The K-m values for the reductants NADPH and NADH over a range of mutants, and for the non-mutant enzyme, differed to a much lesser degree than the K-m for AcPyAD(+) in the two reactions. The K-m(AcPyAD) values for the transhydrogenation of AcPyAD(+) by NADH were over an order of magnitude greater than those for the transhydrogenation of AcPyAD(+) by NADPH, It is unlikely that AcPyAD(+) binds at the same site in both reactions. A plausible explanation is that this substrate binds to the NADP(H)-binding site for transhydrogenation by NADH. Thus, a hydride equivalent can be transferred directly between NADH and AcPyAD(+) under these conditions.