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 _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.