MODIFICATION OF THE ELECTROSTATIC ENVIRONMENT IS TOLERATED IN THE OXYANION HOLE OF THE CYSTEINE PROTEASE PAPAIN

The oxyanion hole in cysteine and serine proteases can be viewed as an arrangement of prealigned dipoles that complements the changes in charge distribution during the enzymatic reaction. Because of the electrostatic nature of the interaction involved in the oxyanion hole, the introduction of charged residues in that region could have a major effect on the catalytic properties of the enzyme. In this study, residue Gln19, which contributes to one of the hydrogen bonds in the oxyanion hole of papain, has been replaced by glutamic acid, histidine, and asparagine residues. These mutations result in 65-315-fold decreases in k(cat)/K-M, supporting our previous finding, that the side chain of Gln19 contributes to transition state stabilization in the oxyanion hole of papain (Menard et al., 1991a). Since papain is active over a wide range of pH values, the influence of side chain ionization on activity could be measured quantitatively with the mutant Gln19Glu. The pH dependency of k(cat)/K-M for Gln19Glu is not of the classical bell-shaped form normally observed for papain, but instead is modulated by ionization of the Glu19 side chain with a pK(a) of 6.02. The Gln19Glu mutant at low pH, where the Glu19 side chain is neutral, is the enzyme that displays activity closest to that of wild-type enzyme, with a (k(cat)/K-M)(1)(lim) value only 20-fold lower than that for papain. As expected, the activity of the Gln19Glu mutant decreases when the Glu19 side chain ionizes. However, introduction of the negatively charged glutamate into the oxyanion hole of papain leads to a further reduction in activity of only 12-fold, and this mutant is still more active than the Gln19Ser enzyme and only 3-fold less active than Gln19Asn. Mutation of Gln19 to His caused the strongest decrease in k(cat)/K-M of the three mutants, a possible consequence of conformational and ion pair stability perturbations. Contrary to the Gln19Glu enzyme, ionization of His19 could not be detected unambiguously. Probable side chain orientations in the mutants were obtained from molecular modeling experiments, and factors affecting the electrostatic environment of the active site of papain are discussed. Comparison of our results with those obtained for a number of subtilisin mutants indicates that transition state stabilization through oxyanion hole interactions might be more important in serine proteases than in cysteine proteases by approximately 0.5-1.0 kcal/mol.