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Mechanistic Imperatives for Deprotonation of Carbon Catalyzed by Triosephosphate Isomerase: Enzyme-Activation by Phosphite Dianion.

ABSTRACT: The mechanistic imperatives for catalysis of deprotonation of ?-carbonyl carbon by triosephosphate isomerase (TIM) are discussed. There is a strong imperative to reduce the large thermodynamic barrier for deprotonation of carbon to form an enediolate reaction intermediate; and, a strong imperative for specificity in the expression of the intrinsic phosphodianion binding energy at the transition state for the enzyme-catalyzed reaction. Binding energies of 2 and 6 kcal/mol, respectively, have been determined for formation of phosphite dianion complexes to TIM and to the transition state for TIM-catalyzed deprotonation of the truncated substrate glycolaldehyde [T. L. Amyes, J. P. Richard, Biochemistry2007, 46, 5841]. We propose that the phosphite dianion binding energy, which is specifically expressed at the transition state complex, is utilized to stabilize a rare catalytically active loop-closed form of TIM. The results of experiments to probe the role of the side chains of Ile172 and Leu232 in activating the loop-closed form of TIM for catalysis of substrate deprotonation are discussed. Evidence is presented that the hydrophobic side chain of Ile172 assists in activating TIM for catalysis of substrate deprotonation through an enhancement of the basicity of the carboxylate side-chain of Glu167. Our experiments link the two imperatives for TIM-catalyzed deprotonation of carbon by providing evidence that the phosphodianion binding energy is utilized to drive an enzyme conformational change, which results in a reduction in the thermodynamic barrier to deprotonation of the carbon acid substrate at TIM compared with the barrier for deprotonation in water. The effects of a P168A mutation on the kinetic parameters for the reactions of whole and truncated substrates are discussed.

SUBMITTER: Zhai X

PROVIDER: S-EPMC3979633 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Mechanistic Imperatives for Deprotonation of Carbon Catalyzed by Triosephosphate Isomerase: Enzyme-Activation by Phosphite Dianion.

Zhai Xiang X Malabanan M Merced MM Amyes Tina L TL Richard John P JP

Journal of physical organic chemistry 20140401 4

The mechanistic imperatives for catalysis of deprotonation of α-carbonyl carbon by triosephosphate isomerase (TIM) are discussed. There is a strong imperative to reduce the large thermodynamic barrier for deprotonation of carbon to form an enediolate reaction intermediate; and, a strong imperative for specificity in the expression of the intrinsic phosphodianion binding energy at the transition state for the enzyme-catalyzed reaction. Binding energies of 2 and 6 kcal/mol, respectively, have been ...[more]

PMID: 24729658

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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:Product yields for the reactions of (R)-glyceraldehyde 3-phosphate (GAP) in D2O at pD 7.9 catalyzed by wildtype triosephosphate isomerase from Trypanosoma brucei brucei (Tbb TIM) and a monomeric variant (monoTIM) of this wildtype enzyme were determined by (1)H NMR spectroscopy and were compared with the yields determined in earlier work for the reactions catalyzed by TIM from rabbit and chicken muscle [O'Donoghue, A. C., Amyes, T. L., and Richard, J. P. (2005), Biochemistry 44, 2610 - 2621]. Three products were observed from the reactions catalyzed by TIM: dihydroxyacetone phosphate (DHAP) from isomerization with intramolecular transfer of hydrogen, d-DHAP from isomerization with incorporation of deuterium from D2O into C-1 of DHAP, and d-GAP from incorporation of deuterium from D2O into C-2 of GAP. The yield of DHAP formed by intramolecular transfer of hydrogen decreases from 49% for the muscle enzymes to 40% for wildtype Tbb TIM to 34% for monoTIM. There is no significant difference in the ratio of the yields of d-DHAP and d-GAP for wildtype TIM from muscle sources and Trypanosoma brucei brucei, but partitioning of the enediolate intermediate of the monoTIM reaction to form d-DHAP is less favorable ((k(C1))(D)/(k(C2))(D) = 1.1) than for the wildtype enzyme ((k(C1))(D)/(k(C2))(D) = 1.7). Product yields for the wildtype Tbb TIM and monoTIM-catalyzed reactions of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-(13)C]-GA) at pD 7.0 in the presence of phosphite dianion and in its absence were determined by (1)H NMR spectroscopy [Go, M. K., Amyes, T. L., and Richard, J. P. (2009) Biochemistry 48, 5769-5778]. There is no detectable difference in the yields of the products of wildtype muscle and Tbb TIM-catalyzed reactions of [1-(13)C]-GA in D2O. The kinetic parameters for phosphite dianion activation of the reactions of [1-(13)C]-GA catalyzed by wildtype Tbb TIM are similar to those reported for the enzyme from rabbit muscle [Amyes, T. L. and Richard, J. P. (2007) Biochemistry 46, 5841-5854], but there is no detectable dianion activation of the reaction catalyzed by monoTIM. The engineered disruption of subunit contacts at monoTIM causes movement of the essential side chains of Lys-13 and His-95 away from the catalytic active positions. We suggest that this places an increased demand that the intrinsic binding energy of phosphite dianion be utilized to drive the change in the conformation of monoTIM back to the active structure for wildtype TIM.

Dataset Information

Mechanistic Imperatives for Deprotonation of Carbon Catalyzed by Triosephosphate Isomerase: Enzyme-Activation by Phosphite Dianion.

Publications

Mechanistic Imperatives for Deprotonation of Carbon Catalyzed by Triosephosphate Isomerase: Enzyme-Activation by Phosphite Dianion.

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