Triosephosphate isomerase as a mechanistic model for utilization of binding energy in enzyme catalysis: Activation and inhibition studies
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Triosephosphate isomerase (TIM) has been demonstrated to be a prototypical example of enzymes utilizing substrate intrinsic binding energy to promote transition state stabilization of the catalyzed reactions. Values of ( k cat / K m ) GAP for TIM-catalyzed reactions of ( R )-glyceraldehyde 3-phosphate and k cat / K HPi K GA for reactions of the substrate pieces glycolaldehyde and HPO 3 2- have been determined for wildtype and more than 10 different strategically designed mutants. A linear logarithmic correlation, with a slope of 1.04 ± 0.03, indicates that the transition states for the two sets of reactions are stabilized by similar interactions with the protein catalyst. This is consistent with a role for dianions as active spectators that hold TIM in a catalytically active caged form. Using phosphite activation as a mechanistic probe, it is possible to dissect the effect of certain mutations into two aspects: the effect on the reactivity of the active conformation of TIM, and the effect on the utilization of the phospho-intrinsic binding energy for catalysis. We have shown that the protein has an architecture that separates the dianion activation site and the catalytic site, so that it is possible to perturb one while leaving the other intact. We also studied the effects of these mutations on the pH-dependence of phosphoglycolate (PGA) inhibition of TIM-catalyzed isomerization of GAP, which allows us to determine the effect of the mutations on the basicity of the catalytic base Glu165/167 in the active conformation of TIM. The correlation between the second-order rate constants of GAP and the inhibition constants of PGA shows that the binding of PGA to TIM mimics the structure of the transition state of the TIM- catalyzed reaction of GAP.