Active-Site Glu165 Activation in Triosephosphate Isomerase and Its Deprotonation Kinetics.
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ABSTRACT: 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 p Ka 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 p Ka. Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the p Ka elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the ?s to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ("heavy enzyme") [U-13C,-15N]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) ?1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of ?s time scale is assigned to temperature-induced p Ka decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ?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.
SUBMITTER: Deng H
PROVIDER: S-EPMC6579534 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
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