The mechanism of hydride transfer between NADH and 3-acetylpyridine adenine dinucleotide by the pyridine nucleotide transhydrogenase of Escherichia coli

Abstract The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD + and NADP + coupled to translocation of protons across the cytoplasmic membrane. Recently, transhydrogenation of 3-acetylpyridine adenine dinucleotide (AcPyAD + ), an analog of NAD + , by NADH has been described using a solubilized preparation of E. coli transhydrogenase [Hutton, M., Day, J.M., Bizouarn, T., and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041–1051]. This reaction depended on the presence of NADP(H). We show that (a) this reaction did not require NADP(H) at pH 6 in contrast to pH 8; (b) the reaction occurred at pH 8 in the absence of NADP(H) in the mutant βH91K and in a mutant in which six amino acids of the carboxy-terminus of the α subunit has been deleted; (c) the mutant transhydrogenases contained bound NADP + and were in a conformation in which the β subunit was digestible by trypsin; (d) the conformation of the β subunit of the wild-type enzyme was made susceptible to trypsin digestion by NADP(H) or by placing the enzyme at pH 6 in the absence of NADP(H). It is concluded that reduction of AcPyAD + by NADH does not involve NADPH as an intermediate and that the role of NADP(H) in this reaction at pH 8 is to cause the transhydrogenase to adopt a conformation favouring transhydrogenation between NADH and AcPyAD + .