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

Author:
Deng, Hua, Dyer, R. Brian, Callender, Robert
Source:
TheJournal of physical chemistry 2019 v.123 no.19 pp. 4230-4241
ISSN:
1520-5207
Subject:
Fourier transform infrared spectroscopy, active sites, carbon, catalytic activity, deprotonation, dissociation, enzyme kinetics, glyceraldehyde 3-phosphate, hydrophilicity, isotopes, moieties, pH, phosphates, triose-phosphate isomerase, yeasts
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 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 Glu165 in the μs 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 μs time scale is assigned to temperature-induced pKₐ 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.
Agid:
6445012