MOLECULAR-ORBITAL STUDIES OF THE STRUCTURE AND REACTIVITY OF MODEL SUBSTRATE INTERMEDIATES IN THE DEACYLATION OF THE CYSTEINE PROTEASE PAPAIN

Molecular orbital calculations suggest that an interaction between the substrate amide nitrogen of substituted N-benzoylglycine (dithioacyl) papains and the thiol sulfur of cysteine-25 is not only catalytically important, it is part of the catalytic mechanism. AM1 indicates that, during hydrolysis of the dithio ester intermediate, charge is exchanged via orbital interactions and in particular between the HOMOs associated with the reaction site and the LUMOs associated with the benzamide portion of the substrate. Charge delocalization emanating from this effect stabilizes the anionic tetrahedral intermediate formed during deacylation, thereby lowering its activation energy and providing the catalytic effect. The unexpected strong effect of electron-withdrawing and -donating groups (located on the substrate's benzamide phenyl) on deacylation kinetics is explained in terms of their ability to promote or inhibit this charge delocalization. The present computational results together with previous spectroscopic studies suggest that the enzyme specifically promotes this interaction in its binding of the substrate. The relative reactivities of dithio esters and thiol esters are contrasted in terms of their electronic features. Both AM1 and ab initio results indicate a reversal of bond polarity for the C=S bond in the dithio ester moiety [C(=S)-S] relative to the C=O bond in the thiol ester moiety [C(=O)-S]. The approximately 20-fold higher rate of deacylation for the thiol ester relative to the dithio ester may in part be accounted for by the increased stability of the tetrahedral intermediate for the dithio ester relative to the thiol ester.