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

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·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 p K_a for deprotonation of EH·I₃^-. The linear free energy correlation, with slope of 0.73 ( r² = 0.96), between k_cat/ K_m 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 p K_a = 10-10.5 were estimated for deprotonation of EH·I₃^- for wildtype TIM. This p K_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 p K_a 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.