Molecular modeling of substrate-enzyme reactions for the cysteine protease papain

AM1 quantum mechanical reaction coordinate (RC) calculations were run to simulate the rate-limiting deacylation (hydrolysis) reaction for a series of para-X-PhC(O)NHCH2-C(Y)-S-papain intermediates, where X = OCH3, CH3, H, Cl, NO2 and Y = O (thioester) or S (dithioester), for which a large body of structural kinetic, and spectroscopic data is available. Several reaction zones, in particular the so-designated Large Zone and Small Zone, were extracted for these RC simulations from the fully solvated and energy-minimized X-ray crystal structure of papain (pdb9pap) bound to the appropriate substrate moiety. The major structural difference between these two zones was rite absence of the oxyanion hole in the latter. For both the thioester and dithioester cases, the calculated E(alpha) value associated with the parent (X = H) acyl-enzyme intermediate was lower by ca. 10 kcal/mol for the Large Zone than for the Small Zone. The magnitude of this difference suggests that the oxidation hole plays a functional if not essential role in stabilizing the anionic tetrahedral intermediate with the cystein proteases. The calculated E(alpha) value was lower by ca. 10 kcal/mol for the thioester [-C(O)-S-] than for the corresponding dithioester [-C(S)-S-], in qualitative agreement with kinetic data for this series of substrates which reveal that the specific rate constant for deacylation k(3) is ca. 60 times larger for the former. This difference is also consistent with both AM1 and 6-31G* calculations on model intermediates, which indicate that the weaker polarity of the dithioester compared with the thioester [i.e., -C(<--S)-S- versus -C(-->O)-S-] renders the former a much poorer site for nucleophilic attack. The anionic tetrahedral intermediate is energetically more stable for the dithioester than for the corresponding thioester, a finding that is discussed in terms of its kinetic and mechanistic implications. The mode of attack by the H2O nucleophile is ''concerted'' rather than ''sequential'' in terms of the order of proton proton abstraction by His-159 and nucleophilic attack on the acyl-enzyme intermediate. While the presumably key S-thiol ... N nonbonded contact remained almost constant (ca. 2.90 Angstrom) up to formation of the [TS] structure, the substrate torsion angles phi and psi rotated significantly as the hybridization around the reaction sire transforms from sp(2) to sp(3) during formation of the tertahedral intermediate. The AM1-calculated frontier molecular orbitals for model thioester and dithioester acyl-enzyme intermediates generally associate the HOMOs with the reaction site and the LUMOs with the benzamide moiety. Computer graphics images corroborate our view that, in relation ro the S-thiol ... N interaction, the HOMOS and LUMOs should be identified, respectively, with S-thiol and N rather than the reverse, as suggested by other workers.