Effects of protein glycosylation on catalysis: Changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction
Effects of protein glycosylation on catalysis: Changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction
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DOI：
10.1021/bi962492r
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发表时间：
1997-03-04
期刊：
影响因子：
2.9
通讯作者：
Klinman, JP
中科院分区：
文献类型：
--
作者：
Kohen, A;Jonsson, T;Klinman, JP
葡萄糖氧化酶的三种糖型，它们的糖基化程度和所产生的相对分子质量不同，它们的催化性质已经被表征。以2-脱氧葡萄糖为研究对象，测量了[1-H-2]-2-脱氧葡萄糖的竞争WT和D/T动力学同位素效应的温度依赖性和活化热。对于136、155和205 kDa的糖型，D/T同位素效应对Arrhenius指数前因子(A(D)/A(T))的影响分别为1.47(+/-0.09)、1.30(+/-0.10)和0.89(+/-0.04)。136 kDa糖型的计算值远高于半经典(无隧道)反应的预期范围(上限为1.22)。异常的A(D)/A(T)通过扩展隧道使之合理化。136、155和205 kDa糖型的活化热分别为8.1(+/-0.4)、11.0(+/-0.3)和13.7(+/-0.3)kcal/mol。显然，较少的糖基化会导致较多的隧道效应和较低的活化热。晶体结构、动力学分析等研究表明，酶的活性部位不受糖基化程度的影响。因此，糖型之间的差异表明，酶多糖被膜的变化导致氢转移步骤的性质发生显著变化，这表明蛋白质表面效应动态地传递到活性部位。
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 WT and D/T kinetic isotope effects and the enthalpy of activation using [1-H-2]-2-deoxyglucose. The D/T isotope effect on the Arrhenius preexponential factor (A(D)/A(T)) 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.