Finally, we note that the curious result that E97D and E97Q substitutions at TIM from Plasmodium falciparum (PfTIM) result in 100-and 4000-fold decreases in kcat for PfTIMcatalyzed isomerization of GAP that are not modeled by our simulations of yTIM. Schwans and coworkers have proposed an explanation for this organismal dependence in the measured kinetics, based on small differences in the structures of TbbTIM and PfTIM, for the larger effect of E97D and E97Q substitutions on kcat for catalysis by PfTIM compared with TbbTIM. Substitution of P166. The triad of nonpolar side chains of P166, I170 and L230 line the active-site cavity of TIM. The observation of falloffs in catalytic activity for P166A, I170A, L230A, and I170A/L230A variants of TIM show that these side chains play a structural role in the enhancement of the reactivity of the carboxylate side chain of E165 for deprotonation of enzyme-bound substrate. The effects of I170A, L230A and I170A/L230A substitutions on the reaction activation barrier reflect global changes in the interactions between polar active-site groups and the enediolate-like transition state observed upon substitution of the hydrophobic side chains of I170A and L230A by water at the variant enzymes. We have previously used the EVB-method successfully to model the effects of I170A, L230A and I170A/L230A substitutions. Wierenga and coworkers have provided a detailed description of the role of the P166 side chain in driving the carboxylate side chain of E165 into a hydrophobic cage, where it is flanked by the hydrophobic side chains of I170 and L230 and lies proximal to the carbon acid substrate. They showed that the X-ray crystal structure of the P166A variant complexed to phosphoglycolate (PDB ID: 2J24 ) is essentially superimposable on the wild-type complex to DHAP (PDB ID: 1NEY ), except that relief of the steric clash between the carbonyl oxygen of G209 and the pyrolidine side chain of P166 at the P166A variant allows the carboxylate side chain of E165 to relax to the "swung-out" position observed for unliganded TIM (Figure ). This small structural change observed in the P166A variant is associated with ca. 50-fold decreases in kcat and 2-fold decreases in Km for the turnover of GAP and DHAP. 81 EVB simulations of the P166A variant were carried out, starting with the crystal structure of the yTIM•DHAP complex (PDB ID: 1NEY ), and substituting a -CH3 group for the P166 side chain. The carboxylate side chain of E165 lies initially in the "swung-in" position for wild-type TIM, but, despite starting with the wild-type crystal structure, these simulations remarkably show a similar relaxation in the position of this side chain to the "swung-out" position observed for the crystal structure of the P166A variant complexed to PGA (Figure ). These changes in structure result in small increases in the enzyme-substrate donor-acceptor distance from 3.0 Å for the complexes between wild-type TIM and DHAP or GAP, to 3.3 Å and 3.1 Å for complexes between the P166A variant of TIM and DHAP (Table ) or GAP (Table ), respectively.