Enzyme Architecture: Amino Acid Side-Chains That Function To Optimize the Basicity of the Active Site Glutamate of Triosephosphate Isomerase

[Image: see text] We report pH rate profiles for k(cat) and K(m) 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 K(i) for inhibition of this reaction by phosphoglycolate trianion (I(3–)). The pH profiles for K(i) show that the binding of I(3–) to TIM (E) to form EH·I(3)(–) 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(–)·I(3)(–) at high pH, in which cases the pH profiles define the pK(a) for deprotonation of EH·I(3)(–). The linear free energy correlation, with slope of 0.73 (r(2) = 0.96), between k(cat)/K(m) for TIM-catalyzed isomerization and the disassociation constant of PGA trianion for TIM shows that EH·I(3)(–) and the transition state are stabilized by similar interactions with the protein catalyst. Values of pK(a) = 10–10.5 were estimated for deprotonation of EH·I(3)(–) for wildtype TIM. This pK(a) decreases to as low as 6.3 for the severely crippled Y208F mutant. There is a correlation between the effect of several mutations on k(cat)/K(m) and on pK(a) for EH·I(3)(–). 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.