title: 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 beta H91K and in a mutant in which six amino acids of the carboxy-terminus of the alpha subunit had been deleted; (c) the mutant transhydrogenases contained bound NADP+ and were in a conformation in which the beta subunit was digestible by trypsin; (d) the conformation of the beta 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+.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
authors.forename: N A
authors.initials: NA
authors.lastname: Glavas
authors.forename: P D
authors.initials: PD
authors.lastname: Bragg
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D009243:NAD; D009249:NADP; D006859:Hydrogen; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases; D014357:Trypsin