MECHANISTIC DEDUCTIONS FROM ISOTOPE EFFECTS IN MULTIREACTANT ENZYME MECHANISMS
MECHANISTIC DEDUCTIONS FROM ISOTOPE EFFECTS IN MULTIREACTANT ENZYME MECHANISMS
复制标题
DOI：
10.1021/bi00510a013
复制
发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
CLELAND, WW
中科院分区：
文献类型：
--
作者：
COOK, PF;CLELAND, WW
在有两个或两个以上底物的酶机制中，当每个底物浓度变化时，通过比较同位素对Vmax和表观V/K值的影响，可以推断动力学机制，并获得底物与酶解离的相对速率的定量信息，而不是通过反应得到产物。还提出了利用其他反应物对由平衡微扰法确定的表观同位素效应的影响来确定相同信息的理论。在肝醇脱氢酶的作用下，DPN从E-DPN-环己醇复合体中释放的速度不是很快，而环己醇的释放速度比其反应生成产物的速度要快得多，因此机理似乎是有序的。对于DPNH和环己酮，反应是随机的，因为DPNH可以有限速度从三元络合物中释放出来。对于酵母醇脱氢酶，丙酮存在时，阻止DPNH从酶中释放，因此机理看起来是有序的，但2-丙醇和DPN从E-DPN-2-丙醇中以相同的速度释放，因此反应在这个方向上是随机的。
In an enzymatic mechanism with 2 or more substrates, comparison of the isotope effects on the Vmax and on the apparent V/K values when each substrate concentration is varied allows one to deduce the kinetic mechanism and obtain quantitative information on the relative rates at which substrates dissociate from the enzyme, as opposed to undergoing reaction to give products. Theory is also presented for using the effects of other reactants on the apparent isotope effects determined by the equilibrium perturbation method to determine the same information. With liver alcohol dehydrogenase, DPN is not released at an appreciable rate from the E-DPN-cyclohexanol complex, while cyclohexanol is released much more rapidly than it reacts to give products, so that the mechanism appears ordered. With DPNH and cyclohexanone, the reaction is random since DPNH can be released from the ternary complex at a finite rate. With yeast alcohol dehydrogenase, acetone when present, prevents DPNH release from the enzyme so that the mechanism appears ordered but 2-propanol and DPN are released at equal rates from E-DPN-2-propanol so that the reaction is random in this direction.