The Effect of pH and Temperature on the Kinetic Parameters of Phosphoglucose Isomerase: PARTICIPATION OF HISTIDINE AND LYSINE IN A PROPOSED DUAL FUNCTION MECHANISM

Abstract

Abstract Phosphoglucose isomerase (d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) from rabbit skeletal muscle has been subjected to a detailed kinetic study of the effect of pH and temperature on the Michaelis constants, the maximal velocities, the dissociation constant for the competitive inhibitor 6-phosphogluconate, and the equilibrium constant of the reaction. A coupled pH-stat assay was established as being suitable for measurement of initial velocities in the forward direction between pH 6.0 and 10.0; analogously, the validity of a coupled spectrophotometric assay of the reverse reaction was demonstrated for the range of pH 6.0 to 9.5. Graphical and computer analysis of Km and Vmax data, obtained with these assays as a function of pH, suggest the participation in isomerase catalysis of two ionizable groups in the enzyme with average pK values of 6.75 and 9.3, at 30°. From the change in pK with temperature, values of 7,700 and 16,000 cal per mole, respectively, have been calculated for the heats of ionization for these two groups. These data are considered to be consistent with the involvement of both an imidazole group of histidine and an e-amino group of lysine. Analysis of Ki data for 6-phosphogluconate, obtained as a function of pH for the forward and the reverse reaction, showed no pK at alkaline pH; this has been interpreted as suggesting that the e-amino group of lysine is not involved in the binding of 6-phosphogluconate to the enzyme. On the basis of this evidence, a dual function mechanism is proposed in which the protonated e-amino nitrogen first catalyzes the opening of the hexose ring structure to yield the straight chain form of hexose 6-phosphate, which then undergoes isomerization via formation of an enediol intermediate, with the nonprotonated nitrogen of the imidazole group participating as a base catalyst in this second step.