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-j for inhibition of this reaction by phosphoglycolate trianion (I3-) The pH profiles for K-i show that the binding of I3- to TIM (E) to form EH center dot 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-center dot I-3(-) at high pH, in which cases the pH profiles define the pK(a) for deprotonation of EH center dot 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 center dot 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 center dot 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 center dot 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 5211, which serve to clamp loop 6 over the substrate; 1170, 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.