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Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase.

ABSTRACT: Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (I170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild-type TIM and its I170A, L230A, and I170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

SUBMITTER: Kulkarni YS

PROVIDER: S-EPMC5543394 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase.

Kulkarni Yashraj S YS Liao Qinghua Q Petrović Dušan D Krüger Dennis M DM Strodel Birgit B Amyes Tina L TL Richard John P JP Kamerlin Shina C L SCL

Journal of the American Chemical Society 20170719 30

Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (I170 and L230), over the ...[more]

PMID: 28683550

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Project description:Kinetic parameters are reported for the reactions of whole substrates (kcat/Km, M(-1) s(-1)) (R)-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) and for the substrate pieces [(kcat/Km)E·HPi/Kd, M(-2) s(-1)] glycolaldehyde (GA) and phosphite dianion (HPi) catalyzed by the I172A/L232A mutant of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM). A comparison with the corresponding parameters for wild-type, I172A, and L232A TbbTIM-catalyzed reactions shows that the effect of I172A and L232A mutations on ?G(?) for the wild-type TbbTIM-catalyzed reactions of the substrate pieces is nearly the same as the effect of the same mutations on TbbTIM previously mutated at the second side chain. This provides strong evidence that mutation of the first hydrophobic side chain does not affect the functioning of the second side chain in catalysis of the reactions of the substrate pieces. By contrast, the effects of I172A and L232A mutations on ?G(?) for wild-type TbbTIM-catalyzed reactions of the whole substrate are different from the effect of the same mutations on TbbTIM previously mutated at the second side chain. This is due to the change in the rate-determining step that determines the barrier to the isomerization reaction. X-ray crystal structures are reported for I172A, L232A, and I172A/L232A TIMs and for the complexes of these mutants to the intermediate analogue phosphoglycolate (PGA). The structures of the PGA complexes with wild-type and mutant enzymes are nearly superimposable, except that the space opened by replacement of the hydrophobic side chain is occupied by a water molecule that lies ?3.5 Å from the basic side chain of Glu167. The new water at I172A mutant TbbTIM provides a simple rationalization for the increase in the activation barrier ?G(?) observed for mutant enzyme-catalyzed reactions of the whole substrate and substrate pieces. By contrast, the new water at the L232A mutant does not predict the decrease in ?G(?) observed for the mutant enzyme-catalyzed reactions of the substrate piece GA.

Project description:Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170-173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186-3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12-13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-(13)C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-(13)C,2-(2)H]GA.

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)).

Project description:We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a approximately 50-fold increase in K(m) for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K(i) for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group; (2) a 12000-fold decrease in k(cat) for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state; and (3) a 6 x 10(5)-fold decrease in k(cat)/K(m), corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D(2)O were quantified as dihydroxyacetone phosphate (DHAP) (27%), [1(R)-(2)H]DHAP (23%), [2(R)-(2)H]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of the transfer of a proton to the TIM-bound enediol(ate) intermediate in D(2)O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a >or=14-fold decrease in k(cat)/K(m) for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-(13)C]GA in D(2)O is [1-(13)C,2,2-di-(2)H]GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k(cat)/K(m) for the reactions of both GAP and the neutral truncated substrate [1-(13)C]GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".

Dataset Information

Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase.

Publications

Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase.

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