A Molecular Dynamics Study of the Stability of Chymotrypsin Acyl Enzymes

We have investigated the enantioselectivity observed in the deacylation rates of a β-substituted β-phenylpropionyl chymotrypsin acyl enzyme by molecular dynamics simulations. The ca. 60-fold difference in deacylation rates for the R and S esters (k_d(S) = 0.17 min⁻¹; k_d(R) = 0.0029 min⁻¹) is presumed to derive from less optimal stabilization of reactive intermediates in the case of the R enantiomer. Structures for both R and S acyl enzymes were constructed starting from the X-ray structure of chymotrypsin phenylethaneboronic acid complex. The ester carbonyl oxygen was placed in the oxyanion binding hole (backbone NH's from Gly-193 and Ser-195), and the phenyl group was placed in the hydrophobic cleft. Structures corresponding to acyl enzymes in which the β-substituent, an acetonyl chain, was engaged in hydrogen bonding with seven possible hydrogen bond donors near the active site were generated. Only three of these (Met-192 NH, Gly-216 NH, and Ser-218 OH) survived rigorous molecular mechanics minimization, and these three structures, in both the R and S acyl enzyme series, were subjected to molecular dynamics. In all three R acyl enzymes, the crucial hydrogen interaction between the ester carbonyl oxygen and the oxyanion binding hole was weakened during the dynamics run, whereas in two of the three S acyl enzymes, these hydrogen bonds persisted. There was also some rearrangement of the hydrogen-bonding configuration for the acetonyl side chain. An additional molecular dynamics study was done on the tetrahedral intermediates corresponding to the deacylation of the R and S acyl enzymes. Although, as expected, the hydrogen-bonding interaction between the oxyanion in the tetrahedral intermediate and the hydrogen bond donors in the oxyanion binding hole was strengthened for both enantiomers, the R enantiomer showed less ideal stabilization. These results provide a rationalization for the deacylation enantioselectivity of these acyl enzymes: The reduced deacylation rate of the R enantiomer could arise from less effective hydrogen-bonding stabilization of both the ester carbonyl in the acyl enzyme and the oxyanion in the deacylation tetrahedral intermediate by the oxyanion binding hole site; the S enantiomer, which preserves excellent hydrogen bond distances and geometry at both stages, is more ideally set up for the attack of water that leads to deacylation.