As hypothesized, the Active oxyanion hole structure provides substantial stabilization (Figure ). Val51 is the predominant stabilizing component during oxyanion formation (TS2), with a favorable interaction energy of -14.7 kcal/mol. Contributions from Gly52 and Leu85 are much smaller (-4.0 kcal/mol and -2.6 kcal/mol, respectively). Surprisingly, when the oxyanion species breaks down in TS4, Val51 no longer interacts with the oxyanion. The N-H shifts direction to interact with the lone pair of electrons on the His178 Nε. To ensure the Active-TS4 geometry optimization was not unintentionally biased by the initial geometry input, the oxyanion strand fragment in the optimized TS4 structure was replaced with that from the optimized TS2 structure. The optimization converged to the same TS4 structure, suggesting this is the appropriate first-order saddle point. Although the Val51 N-H points away from the active site in the Inactive model, the Val51 Cα-H stabilizes TS2 by -5.9 kcal/mol and TS4 by -4.9 kcal/mol (Figure ). Leu85 contributes -4.3 kcal/mol in Inactive-TS2, which is slightly more than its contributions in Active-TS2 and Inactive fGln123-TS2 (Figure ), presumably to compensate for the weak interaction from Val51. However, during oxyanion breakdown in Inactive-TS4, the Gln:Oε-Leu85:H distance extends to 2.59 Å, resulting in no appreciable stabilization.