Molecular modeling of substrate-enzyme reactions for the cysteine protease papain.
Lin Y. | Welsh W.J.
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 the absence of the oxyanion hole in the latter. For both the thioester and dithioester cases, the calculated Ea 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 oxyanion hole plays a functional if not essential role in stabilizing the anionic tetrahedral intermediate with the cysteine proteases. The calculated Ea 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 k3 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(produced by S)-S-versus -C(leads to 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.
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