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

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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Project description:BackgroundSaccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein that shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward reactions of the native-unfolded process. Thermal unfolding scans suggest that no stable intermediates appear in the unfolding of yTIM. However, reported evidence points to the presence of residual structure in the denatured monomer at high temperature.ResultsThermally denatured yTIM showed a clear trend towards the formation of aggregation-prone, β-strand-like residual structure when pH decreased from 8.0 to 6.0, even though thermal unfolding profiles retained a simple monophasic appearance regardless of pH. However, kinetic studies performed over a relatively wide temperature range revealed a complex unfolding mechanism comprising up to three observable phases, with largely different time constants, each accompanied by changes in secondary structure. Besides, a simple sequential mechanism is unlikely to explain the observed variation of amplitudes and rate constants with temperature. This kinetic complexity is, however, not linked to the appearance of residual structure. Furthermore, the rate constant for the main unfolding phase shows small, rather unvarying values in the pH region where denatured yTIM gradually acquires a β-strand-like conformation. It appears, therefore, that the residual structure has no influence on the kinetic stability of the native protein. However, the presence of residual structure is clearly associated with increased irreversibility.ConclusionsThe slow temperature-induced unfolding of yeast TIM shows three kinetic phases. Rather than a simple sequential pathway, a complex mechanism involving off-pathway intermediates or even parallel pathways may be operating. β-strand-type residual structure, which appears below pH 8.0, is likely to be associated with increased irreversible aggregation of the unfolded protein. However, this denatured form apparently accelerates the refolding process.

Project description:Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable in WT KSI.

Project description:The role of the hydrophobic side chains of Ile-172 and Leu-232 in catalysis of the reversible isomerization of R-glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) by triosephosphate isomerase (TIM) from Trypanosoma brucei brucei (Tbb) has been investigated. The I172A and L232A mutations result in 100- and 6-fold decreases in k(cat)/K(m) for the isomerization reaction, respectively. The effect of the mutations on the product distributions for the catalyzed reactions of GAP and of [1-(13)C]-glycolaldehyde ([1-(13)C]-GA) in D(2)O is reported. The 40% yield of DHAP from wild-type Tbb TIM-catalyzed isomerization of GAP with intramolecular transfer of hydrogen is found to decrease to 13% and to 4%, respectively, for the reactions catalyzed by the I172A and L232A mutants. Likewise, the 13% yield of [2-(13)C]-GA from isomerization of [1-(13)C]-GA in D(2)O is found to decrease to 2% and to 1%, respectively, for the reactions catalyzed by the I172A and L232A mutants. The decrease in the yield of the product of intramolecular transfer of hydrogen is consistent with a repositioning of groups at the active site that favors transfer of the substrate-derived hydrogen to the protein or the oxygen anion of the bound intermediate. The I172A and L232A mutations result in (a) a >10-fold decrease (I172A) and a 17-fold increase (L232A) in the second-order rate constant for the TIM-catalyzed reaction of [1-(13)C]-GA in D(2)O, (b) a 170-fold decrease (I172A) and 25-fold increase (L232A) in the third-order rate constant for phosphite dianion (HPO(3)(2-)) activation of the TIM-catalyzed reaction of GA in D(2)O, and (c) a 1.5-fold decrease (I172A) and a larger 16-fold decrease (L232A) in K(d) for activation of TIM by HPO(3)(2-) in D(2)O. The effects of the I172A mutation on the kinetic parameters for the wild-type TIM-catalyzed reactions of the whole substrate and substrate pieces are consistent with a decrease in the basicity of the carboxylate side chain of Glu-167 for the mutant enzyme. The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E(C)) relative to an inactive open form (E(O)).

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

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