Bragg P D
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
FEBS Lett. 1996 Nov 11;397(1):93-6. doi: 10.1016/s0014-5793(96)01147-7.
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 Km 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 Km for AcPyAD+ in the two reactions. The Km(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.
吡啶核苷酸转氢酶是一种质子泵，它催化在NAD⁺和NADP⁺之间等价氢化物离子的可逆转移，同时伴随着质子跨细胞质膜的转运。该酶还催化NADH还原NAD⁺类似物3-乙酰吡啶腺嘌呤二核苷酸（AcPyAD⁺）。有人提出（赫顿等人，（1994年）《欧洲生物化学杂志》219卷，1041 - 1051页），此反应需要NADP(H)作为中间体。因此，结合在转氢酶上NADP(H)结合位点的NADP⁺会被NADH还原，并通过AcPyAD⁺交替结合到NAD(H)结合位点而重新氧化。NADPH对AcPyAD⁺的还原将是NADH对AcPyAD⁺还原过程中的一个部分反应。利用来自在无添加NADP(H)时对NADH将AcPyAD⁺转氢活性升高的突变体的细胞质膜囊泡，比较了NADH和NADPH对AcPyAD⁺还原的动力学。在一系列突变体以及非突变酶中，还原剂NADPH和NADH的米氏常数（Km）值的差异程度，远小于这两种反应中AcPyAD⁺的Km值差异。NADH将AcPyAD⁺转氢的Km(AcPyAD)值比NADPH将AcPyAD⁺转氢的Km(AcPyAD)值大一个数量级以上。两种反应中AcPyAD⁺不太可能结合在同一位点。一个合理的解释是，该底物在NADH转氢反应中结合到NADP(H)结合位点。因此，在这些条件下，一个等价氢化物可以直接在NADH和AcPyAD⁺之间转移。
