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

FEBS Letters

Bragg, Philip D.¹ 

[1] Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada

关键词: Transhydrogenase;    Pyridine nucleotide;    Hydride transfer;    Enzyme mechanism;    Escherechia coli;    AcPyAD+;    3-acetylpyridine adenine dinucleotide;    Mes;    2-[N-morpholino]ethanesulfonic acid;   

DOI  :  10.1016/S0014-5793(96)01147-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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) Eur. 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.

【 授权许可】

Unknown