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

We report pH rate profiles for k and K 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 for inhibition of this reaction by phosphoglycolate trianion (I). The pH profiles for K show that the binding of I to TIM (E) to form EH·I 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 at high pH, in which cases the pH profiles define the p K for deprotonation of EH·I. The linear free energy correlation, with slope of 0.73 ( r = 0.96), between k/ K for TIM-catalyzed isomerization and the disassociation constant of PGA trianion for TIM shows that EH·I and the transition state are stabilized by similar interactions with the protein catalyst. Values of p K = 10-10.5 were estimated for deprotonation of EH·I for wildtype TIM. This p K 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/ K and on p K for EH·I. 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.