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 which show that a substantial fraction of the large 12 kcal/mol intrinsic binding energy of the non-reacting 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 where 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, in order 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.

Mechanism and inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase

“…Thus, 3.2 kcal/mol of the lost transition-state stabilization was recovered in the reaction of ''the substrate in pieces'' [92]. These results agree with those of similar studies involving phosphite activation of reactions with truncated substrates for the mechanistically unrelated enzymes triosephosphate isomerase (TIM) [112], orotidine-5 0 -monophosphate decarboxylase (OMPDC) [113], and glycerol-3 0 -phosphate dehydrogenase (GPDH) [114]. DXR shares one common feature with these enzymes: a flexible loop that closes over the phosphate-binding pocket of the active site in the presence of phosphodianion or an analogue.…”

Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase

“…2,11,51,52 This mechanism is illustrated by Scheme 5 for activation of OMPDC by phosphite dianion, where K C ≪ 1 for the protein conformational change. The open form of OMPDC ( E O ) is disordered, due to the conformational flexibility of the phosphodianion and pyrimidine gripper loops (Figure 1), and the phosphodianion binding energy is utilized, in part, to organize/position the catalytic side chains at the closed enzyme E C .…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…The active site is located at the carboxyl end of the barrel (45,(146)(147)(148). The enzyme follows an enediolate intermediate mechanism (Glu144 [P. woesei numbering] acid/base catalyst) (150,151). His96 stabilizes the intermediate, and a highly conserved and also catalytically essential lysine (Lys14) also fulfills a stabilizing role, interacting with the phosphate group of the substrate.…”