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 gly cosylation and resulting molecular weight, have been characterized wit h 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 act ivation using [1-H-2]-2-deoxyglucose. The D/T isotope effect on the Ar rhenius 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, re spectively. The value obtained for the 136 kDa glycoform is well above the range expected for semiclassical-classical (no tunneling) reactio ns (upper limit of 1.22). The abnormal A(D)/A(T) is rationalized by ex tensive 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 gl ycoforms, 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 sit e is not conformationally changed by the degree of glycosylation. Henc e, the differences among the glycoforms, which indicate that changes i n the enzyme polysaccharide envelope lead to a significant change in t he nature of the hydrogen transfer step, suggest a dynamic transmissio n of protein surface effects to the active site.