Effects of protein glycosylation on catalysis: changes in hydrogen tunneling and enthalpy of activation in the glucose oxidase reaction.

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.