Bizouarn T, Grimley R L, Cotton N P, Stilwell S N, Hutton M, Jackson J B
School of Biochemistry, University of Birmingham, Edgbaston, UK.
Biochim Biophys Acta. 1995 Apr 4;1229(1):49-58. doi: 10.1016/0005-2728(94)00186-9.
Proton-translocating transhydrogenase was solubilised and purified from membranes of Escherichia coli. Consistent with recent evidence [Hutton, M., Day, J., Bizouarn, T. and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051], at low pH and salt concentration, the enzyme catalysed rapid reduction of the NAD+ analogue AcPdAD+ by a combination of NADH and NADPH. At saturating concentrations of NADPH, the dependence of the steady-state rate on the concentrations of NADH and AcPdAD+ indicated that, with respect to these two nucleotides, the reaction proceeds by a ping-pong mechanism. High concentrations of either NADH or AcPdAD+ led to substrate inhibition. These observations support the view that, in this reaction, NADP(H) remains bound to the enzyme: AcPdAD+ is reduced by enzyme-bound NADPH, and NADH is oxidised by enzyme-bound NADP+, in a cyclic process. When this reaction was carried out with [4A-2H]NADH replacing [4A-1H]NADH, the rate was decreased by 46%, suggesting that the H- transfer steps are rate-limiting. In simple 'reverse' transhydrogenation, the reduction of AcPdAD+ was slower with [4B-2H]NADPH than with [4B-1H]NADPH when the reaction was performed at pH 8.0, but there was no deuterium isotope effect at pH 6.0. This indicates that H- transfer is rate-limiting at pH 8.0 and supports our earlier suggestion that NADP+ release from the enzyme is rate-limiting at low pH. The lack of a deuterium isotope effect in the reduction of thio-NADP+ by NADH at low pH is also consistent with the view that NADPH release from the enzyme is slow under these conditions. A steady-state rate equation is derived for the reduction of AcPdAD+ by NADPH plus NADH, assuming operation of the cyclic pathway. It adequately accounts for the pH dependence of the enzyme, for the features described above and for kinetic characteristics of E. coli transhydrogenase described in the literature.
质子转运型转氢酶从大肠杆菌细胞膜中溶解并纯化出来。与最近的证据一致[赫顿，M.，戴，J.，比祖阿尔恩，T.和杰克逊，J.B.（1994年）《欧洲生物化学杂志》219卷，1041 - 1051页]，在低pH值和盐浓度下，该酶通过NADH和NADPH的组合催化NAD +类似物AcPdAD +的快速还原。在NADPH饱和浓度下，稳态速率对NADH和AcPdAD +浓度的依赖性表明，就这两种核苷酸而言，反应通过乒乓机制进行。高浓度的NADH或AcPdAD +会导致底物抑制。这些观察结果支持这样一种观点，即在该反应中，NADP(H)仍与酶结合：AcPdAD +被酶结合的NADPH还原，NADH被酶结合的NADP +氧化，这是一个循环过程。当用[4A - 2H]NADH替代[4A - 1H]NADH进行该反应时，速率降低了46%，这表明氢转移步骤是限速步骤。在简单的“反向”转氢反应中，当反应在pH 8.0下进行时，[4B - 2H]NADPH比[4B - 1H]NADPH还原AcPdAD +的速度慢，但在pH 6.0时没有氘同位素效应。这表明在pH 8.0时氢转移是限速步骤，并支持我们早期的观点，即在低pH值下酶释放NADP +是限速步骤。在低pH值下NADH还原硫代NADP +时缺乏氘同位素效应也与这样一种观点一致，即在这些条件下酶释放NADPH很慢。假设循环途径起作用，推导了NADPH加NADH还原AcPdAD +的稳态速率方程。它充分解释了酶的pH依赖性、上述特征以及文献中描述的大肠杆菌转氢酶的动力学特性。
