Stilwell S N, Bizouarn T, Jackson J B
School of Biochemistry, University of Birmingham, Edgbaston, UK.
Biochim Biophys Acta. 1997 May 16;1320(1):83-94. doi: 10.1016/s0005-2728(97)00016-9.
Transhydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.
转氢酶是一种质子泵。它具有分别用于NAD⁺/NADH（在蛋白质的结构域I上）和NADP⁺/NADPH（在结构域III上）的结合位点。从大肠杆菌中纯化得到的、用去污剂分散的转氢酶，在没有添加NADP⁺或NADPH的情况下，能以缓慢的速率催化NADH还原NAD⁺类似物乙酰吡啶腺嘌呤二核苷酸（AcPdAD⁺）。尽管这个反应很慢，但却令人惊讶，因为转氢酶通常被认为是通过三元复合物机制催化NAD(H)及其类似物与NADP(H)及其类似物之间的氢化物转移。研究表明，氢化物转移发生在NADH烟酰胺环上的4A位与AcPdAD⁺的4A位之间。基于该酶已知的立体特异性，这排除了以下两种转氢的可能性：(a) 从结构域I中的NADH转移到错误定位在结构域III中的AcPdAD⁺；以及(b) 从错误定位在结构域III中的NADH转移到结构域I中的AcPdAD⁺。在添加低浓度的NADP⁺或NADPH时，用去污剂分散的大肠杆菌转氢酶能催化NADH非常快速地还原AcPdAD⁺。这个反应是循环的；它通过AcPdAD⁺氧化NADPH以及NADH还原NADP⁺交替进行，而NADPH和NADP⁺则紧密结合在酶上。在本研究中，表明循环反应的速率以及在没有添加NADP⁺/NADPH时NADH还原AcPdAD⁺的速率，对pH和MgSO₄浓度具有相似的依赖性，并且它们具有相似的动力学特征。因此，有人提出NADH还原AcPdAD⁺实际上是一个循环反应，要么是在一小部分（<5%）酶上与紧密结合的NADP⁺/NADPH一起进行，要么是与非自然地封闭在结构域III位点内的NAD⁺/NADH（或AcPdAD⁺/AcPdADH）一起进行。与红螺菌的膜囊泡（载色体）相关的转氢酶在没有添加NADP⁺/NADPH的情况下也能催化NADH还原AcPdAD⁺。当从载色体上剥离转氢酶结构域I时，该反应与“正常反向”转氢反应（例如，NADPH还原AcPdAD⁺）同时丧失。用重组结构域I蛋白进行重组后，这两个反应完全恢复。然而，在对去除了结构域I的载色体进行反复洗涤后，反向转氢活性（在存在结构域I的情况下进行测定时）得以保留，而NADH还原AcPdAD⁺的活性则下降。添加低浓度的NADP⁺或NADPH总是能支持相同的高NADH→AcPdAD⁺反应速率，而与膜洗涤的次数无关。得出的结论是，与纯化的大肠杆菌酶一样，载色体中NADH还原AcPdAD⁺是一个涉及紧密结合在转氢酶结构域III位点上的核苷酸的循环反应。然而，就红螺菌的膜而言，可以比较确定地表明结合的核苷酸是NADP⁺或NADPH。因此，无需借助转氢酶上存在多个核苷酸结合位点的说法就能充分解释这些数据。
