Kohen A, Jonsson T, Klinman J P
Department of Chemistry, University of California, Berkeley 94720, USA.
Biochemistry. 1997 Mar 4;36(9):2603-11. doi: 10.1021/bi962492r.
Three glycoforms of glucose oxidase, which vary in their degree of glycosylation and resulting molecular weight, have been characterized with regard to catalytic properties. Focusing on 2-deoxyglucose to probe the chemical step, we have now measured the temperature dependence of competitive H/T and D/T kinetic isotope effects and the enthalpy of activation using [1-2H]-2-deoxyglucose. The D/T isotope effect on the Arrhenius preexponential factor (AD/AT) is 1.47 (+/-0.09), 1.30 (+/-0.10), and 0.89 (+/-0.04) for the 136, 155, and 205 kDa glycoforms, respectively. The value obtained for the 136 kDa glycoform is well above the range expected for semiclassical-classical (no tunneling) reactions (upper limit of 1.22). The abnormal A(D)/A(T) is rationalized by extensive tunneling. The enthalpies of activation are 8.1 (+/-0.4), 11.0 (+/-0.3), and 13.7 (+/-0.3) kcal/mol for the 136, 155, and 205 kDa glycoforms, respectively. Apparently, less glycosylation results in more tunneling and a lower enthalpy of activation. The crystal structure, kinetic analysis, and other studies suggest that the enzyme active site is not conformationally changed by the degree of glycosylation. Hence, the differences among the glycoforms, which indicate that changes in the enzyme polysaccharide envelope lead to a significant change in the nature of the hydrogen transfer step, suggest a dynamic transmission of protein surface effects to the active site.
已对葡萄糖氧化酶的三种糖型进行了催化特性表征，这三种糖型在糖基化程度和由此产生的分子量方面存在差异。以2-脱氧葡萄糖为探针研究化学步骤，我们现在使用[1-2H]-2-脱氧葡萄糖测量了竞争性H/T和D/T动力学同位素效应的温度依赖性以及活化焓。对于136 kDa、155 kDa和205 kDa的糖型，D/T同位素对阿仑尼乌斯指前因子（AD/AT）的影响分别为1.47（±0.09）、1.30（±0.10）和0.89（±0.04）。136 kDa糖型获得的值远高于半经典-经典（无隧穿）反应预期的范围（上限为1.22）。异常的A(D)/A(T)可通过广泛的隧穿来解释。对于136 kDa、155 kDa和205 kDa的糖型，活化焓分别为8.1（±0.4）、11.0（±0.3）和13.7（±0.3）kcal/mol。显然，糖基化程度越低，隧穿越多，活化焓越低。晶体结构、动力学分析和其他研究表明，酶活性位点不会因糖基化程度而发生构象变化。因此，糖型之间的差异表明酶多糖包膜的变化导致氢转移步骤性质的显著变化，这表明蛋白质表面效应向活性位点的动态传递。
