Magnitude and origin of the enhanced basicity of the catalytic glutamate of triosephosphate isomerase

Glu-167 of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM) acts as the base to deprotonate substrate to form an enediolate phosphate trianion intermediate. We report that there is a large ∼6 pK unit increase in the basicity of the carboxylate side chain of Glu-167 upon binding of the inhibitor phosphoglycolate trianion (I^3-), an analog of the enediolate phosphate intermediate, from pK_EH ≈ 4 for the protonated free enzyme EH to pK_EHI ≈ 10 for the protonated enzyme-inhibitor complex EH•I^3-. We propose that there is a similar increase in the basicity of this side chain when the physiological substrates are deprotonated by TbbTIM to form an enediolate phosphate trianion intermediate and that it makes an important contribution to the enzymatic rate acceleration. The affinity of wildtype TbbTIM for I^3- increases 20 000-fold upon decreasing the pH from 9.3 to 4.9, because TbbTIM exists mainly in the basic form E over this pH range, while the inhibitor binds specifically to the rare protonated enzyme EH. This reflects the large increase in the basicity of the carboxylate side chain of Glu-167 upon binding of I^3- to EH to give EH•I^3-. The I172A mutation at TbbTIM results in an ∼100-fold decrease in the affinity of TbbTIM for I^3- at pH < 6 and an ∼2 pK unit decrease in the basicity of the carboxylate side chain of Glu-167 at the EH•I^3- complex, to pK_EHI = 7.7. Therefore, the hydrophobic side chain of Ile-172 plays a critical role in effecting the large increase in the basicity of the catalytic base upon the binding of substrate and/or inhibitors.