Active-Site Glu165 Activation in Triosephosphate Isomerase and Its Deprotonation Kinetics

Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a C═O stretch band at 1706 cm–1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the pKa of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased pKa. Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pKa elevation of the catalytic base Glu165. The deprotonation kinetics of Glu16...