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Enzyme Architecture: Amino Acid Side-Chains That Function To Optimize the Basicity of the Active Site Glutamate of Triosephosphate Isomerase
- Zhai, Xiang, Reinhardt, Christopher J., Malabanan, M. Merced, Amyes, Tina L., Richard, John P.
- Journal of the American Chemical Society 2018 v.140 no.26 pp. 8277-8286
- Gibbs free energy, active sites, catalysts, catalytic activity, glutamic acid, glyceraldehyde 3-phosphate, hydrophobicity, isomerization, models, mutants, mutation, pH, triose-phosphate isomerase
- We report pH rate profiles for kcₐₜ and Kₘ for the isomerization reaction of glyceraldehyde 3-phosphate catalyzed by wildtype triosephosphate isomerase (TIM) from three organisms and by ten mutants of TIM; and, for Kᵢ for inhibition of this reaction by phosphoglycolate trianion (I³–). The pH profiles for Kᵢ show that the binding of I³– to TIM (E) to form EH·I₃– is accompanied by uptake of a proton by the carboxylate side-chain of E165, whose function is to abstract a proton from substrate. The complexes for several mutants exist mainly as E–·I₃– at high pH, in which cases the pH profiles define the pKₐ for deprotonation of EH·I₃–. The linear free energy correlation, with slope of 0.73 (r² = 0.96), between kcₐₜ/Kₘ for TIM-catalyzed isomerization and the disassociation constant of PGA trianion for TIM shows that EH·I₃– and the transition state are stabilized by similar interactions with the protein catalyst. Values of pKₐ = 10–10.5 were estimated for deprotonation of EH·I₃– for wildtype TIM. This pKₐ decreases to as low as 6.3 for the severely crippled Y208F mutant. There is a correlation between the effect of several mutations on kcₐₜ/Kₘ and on pKₐ for EH·I₃–. The results support a model where the strong basicity of E165 at the complex to the enediolate reaction intermediate is promoted by side-chains from Y208 and S211, which serve to clamp loop 6 over the substrate; I170, which assists in the creation of a hydrophobic environment for E165; and P166, which functions in driving the carboxylate side-chain of E165 toward enzyme-bound substrate.