Davies D D, Kenworthy P
Biochem J. 1982 Sep 1;205(3):581-4. doi: 10.1042/bj2050581.
The reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the 'B' methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits 'A' stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the 'B' to the 'A' side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is 'B'-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the 'B' side. A further increase in the removal of hydrogen from the 'B' side of NADPH could be the kinetic isotope effect that is observed when ['A'-3H]NADPH is the reductant, the H--C bond being cleaved 2.3 times faster than the 3H--C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a 'B'-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.
由产朊假丝酵母提取物催化的还原型烟酰胺腺嘌呤二核苷酸对硝酸盐的还原作用，对NADPH的“B”亚甲基氢原子表现出明显的高度立体特异性，对NADH的亚甲基氢原子表现出混合立体特异性。另一方面，纯化的硝酸还原酶对NADH和NADPH表现出“A”立体特异性。酶纯化后，NADPH的立体特异性从“B”侧到“A”侧的明显转变，部分原因是在粗提取物中硝酸盐被完全还原为氨。亚硝酸盐不会积累，而是被具有“B”特异性的亚硝酸脱氢酶还原为氨，因此在硝酸盐还原过程中从NADPH去除的氢中，高达75%可能来自“B”侧。当['A'-3H]NADPH作为还原剂时观察到的动力学同位素效应，可能是NADPH“B”侧氢去除进一步增加的原因，其中H—C键的断裂速度比3H—C键快2.3倍。观察到的NADH的混合立体特异性可追溯到一种未鉴定的酶，该酶催化NAD+和NADH之间的“B”特异性交换。讨论了该反应与它可能解释其他已报道的明显混合立体特异性情况的可能性之间的关系。
