Use of a minimum perturbation approach to predict TIM mutant structures.

A minimum perturbation conformational search approach is used to model the structures of the yeast triosephosphate isomerase (TIM) single mutant in which the catalytic base Glu165 is changed to Asp, and the double mutant in which Glu165 is changed to Asp and Ser96 to Pro. In chicken TIM this double mutant is referred to as a pseudo-revertant because some of the catalytic activity lost due to the first mutation is regained when the second mutation occurs. Three minimum energy structures were calculated for the Asp165 conformation in the yeast TIM single mutant and another three for the double mutant. One of the calculated minimum energy conformations for Asp165 in the E165D structure agrees well with the X-ray structure. However, this conformation is not that of the lowest energy and is not one of the three most common conformers for Asp found by Ponder and Richards. This suggests that when an amino acid is introduced it may not be able to conform to the more general rules that apply to protein structures of evolutionary origin. While the van der Waals energy largely determines the allowed minima, the relative ranking of the final minima is determined by electrostatic effects and can therefore be affected by the inclusion of crystal waters in the calculation. When the E165D calculation is repeated with an active-site water molecule fixed in its E165D X-ray structure position, the relative ranking of the minima shifts and the X-ray conformation for Asp165 is the lowest interaction energy conformer. Two of the E165D calculated minimum energy structures are essentially identical to two of the S96P/E165D minima. All of the calculated minima for both the E165D and S96P/E165D mutants position the Asp side chain such that the anti-orbital, and not the more basic syn-orbital, of the carboxylate would be utilized for proton abstraction. This observation may explain why the chicken TIM S96P/E165D mutant, for which the X-ray structure indicates that the syn-orbital is used, is a pseudo-revertant while the yeast TIM double mutant is not; no X-ray structure is available for the latter. The multiplicity of minima found in the present analysis makes clear that predicting the exact orientation of a single side chain is not as simple as might be expected.