Gavish B, Werber M M
Biochemistry. 1979 Apr 3;18(7):1269-75. doi: 10.1021/bi00574a023.
The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements.
已对粘度对羧肽酶A催化速率的影响进行了测试。以三肽苄氧羰基-L-丙氨酰-L-丙氨酰-L-丙氨酸[Z(L-Ala)3]为底物，结果表明，水溶液中存在30%或40%的甲醇或甘油时，对水解速率的大部分影响可归因于粘度对催化速率常数kcat的贡献。在30%和40%的甘油或甲醇中，kcat的阿仑尼乌斯图呈线性且几乎平行。当对不同介质的粘度进行“校正”后，各阿仑尼乌斯图之间的差异显著减小；若同时考虑溶液介电常数的影响，在实验误差范围内差异消失。有人提出，介质的粘度可影响酶促反应的限速步骤，即产物形成前越过能垒的转变速率。根据所提出的机制，酶-底物复合物可通过依赖粘度的结构波动克服这一能垒。理论与实验结果之间的定量一致性表明：(a)由于溶液粘度的温度依赖性，反应的势能垒比观察到的活化能低约5千卡/摩尔；(b)通过动力学测量可获得有关复合物结构灵活性的信息。
