O'Leary M H, Yamada H, Yapp C J
Biochemistry. 1981 Mar 17;20(6):1476-81. doi: 10.1021/bi00509a011.
The enzymatic decarboxylation of glutamic acid shows a carbon isotope effect k12/k13 = 1.018 at 37 degree C, pH 4.7. In D2O under otherwise identical conditions, k12/k13 = 1.009. Under the same conditions solvent isotope effects are Vmax H2O/Vmax D2O = 5.0 and (Vmax/Km)H2O/(Vmax/Km)D2O = 2.6. With the assumption that the carbon isotope effect on the decarboxylation step is in the usual range (1.05--1.07), it is possible to derive relative rates and solvent isotope effects for all steps in the enzyme mechanism. Substrate binding in approximately 2-fold weaker in H2O than in D2O, probably because of the desolvation which accompanies binding of the substrate to the enzyme. A proton inventory analysis of the reaction shows that the Schiff base interchange has a large solvent isotope effect composed of relatively small contributions from at least four separate sites. A conformation change probably accompanies this step. The decarboxylation step shows a solvent isotope effect of approximately 2. Schiff base interchange and decarboxylation are both partially rate determining. The pH dependence of the isotope effects indicates that the initial step in the reaction can occur by way of two different pathways.
谷氨酸的酶促脱羧反应在37℃、pH 4.7时表现出碳同位素效应k12/k13 = 1.018。在其他条件相同的D2O中，k12/k13 = 1.009。在相同条件下，溶剂同位素效应为Vmax H2O/Vmax D2O = 5.0以及(Vmax/Km)H2O/(Vmax/Km)D2O = 2.6。假设碳同位素对脱羧步骤的效应处于通常范围（1.05 - 1.07），则有可能推导出酶促反应机制中所有步骤的相对速率和溶剂同位素效应。底物在H2O中的结合能力比在D2O中弱约两倍，这可能是由于底物与酶结合时伴随的去溶剂化作用。对该反应的质子存量分析表明，席夫碱交换具有较大的溶剂同位素效应，该效应由至少四个不同位点的相对较小贡献组成。此步骤可能伴随着构象变化。脱羧步骤显示出约为2的溶剂同位素效应。席夫碱交换和脱羧反应均部分决定反应速率。同位素效应的pH依赖性表明，反应的初始步骤可以通过两种不同途径发生。
