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A paradigm for enzyme-catalyzed proton transfer at carbon: triosephosphate isomerase.

ABSTRACT: Triosephosphate isomerase (TIM) catalyzes the stereospecific 1,2-proton shift at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate through a pair of isomeric enzyme-bound cis-enediolate phosphate intermediates. The chemical transformations that occur at the active site of TIM were well understood by the early 1990s. The mechanism for enzyme-catalyzed isomerization is similar to that for the nonenzymatic reaction in water, but the origin of the catalytic rate acceleration is not understood. We review the results of experimental work that show that a substantial fraction of the large 12 kcal/mol intrinsic binding energy of the nonreacting phosphodianion fragment of TIM is utilized to activate the active site side chains for catalysis of proton transfer. Evidence is presented that this activation is due to a phosphodianion-driven conformational change, the most dramatic feature of which is closure of loop 6 over the dianion. The kinetic data are interpreted within the framework of a model in which activation is due to the stabilization by the phosphodianion of a rare, desolvated, loop-closed form of TIM. The dianion binding energy is proposed to drive the otherwise thermodynamically unfavorable desolvation of the solvent-exposed active site. This reduces the effective local dielectric constant of the active site, to enhance stabilizing electrostatic interactions between polar groups and the anionic transition state, and increases the basicity of the carboxylate side chain of Glu-165 that functions to deprotonate the bound carbon acid substrate. A rebuttal is presented to the recent proposal [Samanta, M., Murthy, M. R. N., Balaram, H., and Balaram, P. (2011) ChemBioChem 12, 1886-1895] that the cationic side chain of K12 functions as an active site electrophile to protonate the carbonyl oxygen of DHAP.

SUBMITTER: Richard JP

PROVIDER: S-EPMC3319633 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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A paradigm for enzyme-catalyzed proton transfer at carbon: triosephosphate isomerase.

Richard John P JP

Biochemistry 20120320 13

Triosephosphate isomerase (TIM) catalyzes the stereospecific 1,2-proton shift at dihydroxyacetone phosphate (DHAP) to give (R)-glyceraldehyde 3-phosphate through a pair of isomeric enzyme-bound cis-enediolate phosphate intermediates. The chemical transformations that occur at the active site of TIM were well understood by the early 1990s. The mechanism for enzyme-catalyzed isomerization is similar to that for the nonenzymatic reaction in water, but the origin of the catalytic rate acceleration i ...[more]

PMID: 22409228

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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.

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A paradigm for enzyme-catalyzed proton transfer at carbon: triosephosphate isomerase.

Publications

A paradigm for enzyme-catalyzed proton transfer at carbon: triosephosphate isomerase.

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