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 pK(a). Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the pK(a) elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the mu s to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ("heavy enzyme") [U-C-13,-N-15]YeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) similar to 1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of mu s time scale is assigned to temperature-induced pK(a) decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE similar to 0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.