Venning J D, Bizouarn T, Cotton N P, Quirk P G, Jackson J B
School of Biochemistry, University of Birmingham, Edgbaston, UK.
Eur J Biochem. 1998 Oct 1;257(1):202-9. doi: 10.1046/j.1432-1327.1998.2570202.x.
Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD+/NADH, and domain III for NADP+/NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP+ (k approximately 0.03 s(-1)). Phase A of the burst (k approximately 600 s(-1)) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k approximately 10-50 s(-1)), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcPdAD+ reduction by [4B-2H]NADPH, and by [4B-1H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27535-27538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I:III complex.
转氢酶催化还原当量在NAD(H)和NADP(H)之间的转移，此过程与质子跨细菌和线粒体膜的转运相偶联。该蛋白质具有三结构域结构。结构域I和III从膜上突出（如在细菌的细胞质一侧），结构域II跨膜。结构域I有NAD+/NADH的结合位点，结构域III有NADP+/NADPH的结合位点。我们已分别从红螺菌转氢酶中纯化出结构域I和III的重组形式。当将这两种重组蛋白与底物在停流分光光度计中混合时，从NADPH到NAD+类似物乙酰吡啶腺嘌呤二核苷酸（AcPdAD+）有一个双相的氢化物转移猝发。对应于结构域III单次周转的猝发，先于稳态的开始，稳态受产物NADP+非常缓慢的释放（k约为0.03 s-1）限制。猝发的A相（k约为600 s-1）可能源于结构域I和III复合物中快速的氢化物转移。B相（k约为10 - 50 s-1），当结构域I的浓度低于结构域III时占主导，可能是由于结构域I:III复合物的解离以及结构域I的进一步缔合和周转导致的。A相和B相仅对pH有微弱依赖性，因此氢化物转移反应或伴随I:III复合物解离的构象变化不太可能直接与质子结合或释放相偶联。在A相期间，对[4B - 2H]NADPH和[4B - 1H]NADPH还原AcPdAD+的温度依赖性进行比较表明，量子力学隧穿可能对氢化物转移过程有贡献。鉴于核苷酸之间的氢化物转移是直接的[Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27535 - 27538]，这表明在I:III复合物中两个核苷酸的烟酰胺环非常接近。
