Molecular dynamics simulations of active site mutants of triosephosphate isomerase.

Molecular dynamics simulations of triosephosphate isomerase (TIM) and of some active site TIM mutants were performed in an attempt to elucidate possible interactions important for catalytic activity and binding. A variety of active site residues in TIM have been altered, resulting in all cases in decreases in catalytic activity. Second-site suppressor mutants were characterized for two of these active site mutants. The pseudorevertants have increased activity compared to the single mutant from which they were derived and, surprisingly, in both cases the increase in activity is a result of the replacement of an active site serine for proline. We performed simulations of wild-type TIM and the active site mutants with the substrate dihydroxyacetone phosphate bound both noncovalently and covalently. The noncovalent complexes were used to examine interactions important to binding while the covalent complexes are models of the transition state structure for enolization, which is the rate-determining step for the mutants. The difference between these two states, then, is related to the catalytic activity. We found various protein-substrate interactions that improved in the noncovalent mutant complexes, which correlates with the experimentally observed increase in binding affinity upon mutation. In the covalent complexes we observed improved electrostatic stabilization of the transition state upon introduction of Pro, which is also consistent with the experimental data. Our simulations reproduce the highly co-operative nature of the interactions in the active site and suggest that this approach may be useful for identifying particularly promising sites for mutation.