Electrostatic Control Of Enzyme Reactions: Effect Of Ionic Strength On The pKa Of An Essential Acidic Group On Glucose Oxidase

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The dissociation constant of an essential acidic group on the redud form of glucose oxidase from Aspergillus niger K₄ has been found to be extremely sensitive to ionic strength. Increasing the ionic strength from 0.025 to 0.225 causes a decrease in pK4,0w of 0.9 pH unit, from 8.2 to 7.3. Analysis of the ionic strength dependence of pK(4,obsd), making the assumption that the enzyme is a homogeneously charged impenetrable sphere [Edsall, J. T., & Wyman, J. (1958) Biophysical Chemistry, Vol. 1, pp 282-289, 512-514, Academic Press, New York], predicts that the intrinsic pK(g) of the acidic group is 6.7 and that the charge on the protein is -78. The enzyme was titrated from its isoelectric point (pH 4.05) to pH 7.7, the pH at which the ionic strength dependence was determined. It was found to have an actual charge at that pH of -77, in remarkable agreement with the theoretical prediction. Thus, glucose oxidase exerts electrostatic control on pK(4,obsd) as though it were a uniformly charged sphere. The group responsible for pK(4,obsd) has not been identified. However, its measured ΔH°(obsd) of 8.0 kcal mol⁻¹ and ΔS°(obsd) of -6.1 cal mol⁻¹ K⁻¹, together with its pKₐ, of 6.7, are consistent with the group being a histidine residue.

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