Bohren K M, von Wartburg J P, Wermuth B
Institut für Biochemie und Molekularbiologie der Universität Bern, Switzerland.
Biochem J. 1987 May 15;244(1):165-71. doi: 10.1042/bj2440165.
Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. Deuterium isotope effects on the apparent V/Km values decreased with increasing concentrations of menadione but were independent of the NADPH concentration. The results, together with data from product inhibition studies, are consistent with carbonyl reductase obeying a compulsory-order mechanism, NADPH binding first and NADP+ leaving last. No significant differences in the kinetic properties of three molecular forms of carbonyl reductase were detectable.
通过NADPH对羰基还原酶催化的甲萘醌还原反应进行初速率分析，在双倒数图中得到了一系列直线，这与遵循的有序机制一致。在存在羰基还原酶的情况下，NADPH的荧光增加了7倍，同时发射最大值向较低波长移动，并且NADPH和NADP+都导致酶荧光猝灭，表明形成了二元酶-辅酶复合物。随着甲萘醌浓度的增加，氘同位素对表观V/Km值的影响降低，但与NADPH浓度无关。这些结果与产物抑制研究的数据一起，与羰基还原酶遵循强制顺序机制一致，即NADPH首先结合，NADP+最后离开。三种分子形式的羰基还原酶的动力学性质没有检测到显著差异。
