FIGURE 6.

Incursion of potentially hydrolytic water molecules into the active site is not rate-limiting. A, solvent hydration box surrounding the mesotrypsin-APPI substrate complex is shown; zoom-in panel shows the Arg-15 carbonyl carbon (green sphere) with waters that satisfy the 3.5 Å distance criterion at any point throughout the duration of the simulation colored in red. B, representative structure is shown for a water (water 1533) that met geometric criteria for nucleophilic attack geometry, simultaneously approaching within 3.5 Å of the Lys-15 carbonyl C and mesotrypsin His-57(Nϵ2) in the BPTI acyl-enzyme simulation. C, distance versus time plots show entry and residence time for potential hydrolytic waters that met geometric criteria for 50 ns or longer in each simulation. Water O and Ala-16 N positions are plotted as summed distances of these atoms to (i) Lys/Arg-15 carbonyl C and (ii) His-57(Nϵ2).