The ionization behavior of groups at the active site of papain was determined from the pH dependence of the difference of proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by two independent methods. One method involved potentiometric measurements of the protons released and demethylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3 to 7.6 on deprotonation of His-159 at 29 degrees C/20.05. Similarly, the pK of His-159 shifts from 4.3 to 8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate that equilibrium constant for transfer of the proton from Cys-25 to His-159 is 8--12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.

Papain was succinylated in order to increase its solubility above pH 8 so that proton NMR spectroscopy could be used to study the ionization of His-159 at the active site of the enzyme. The pH dependence of NMR spectra of catalytically active succinyl-papain and the methylthio derivative of the active-site cysteinyl residue of succinyl-papain (succinyl-papain-S-SCH3) were determined between pH 6 and 10. The pH dependence of the C epsilon 1 H resonance of His-159 in catalytically active succinyl-papain indicates that His-159 has a pK of about 8.6 in the catalytically active form of the enzyme. The position of this resonance in succinyl-papain-S-SCH3 indicates that when the active-site cysteinyl residue is methylthiolated, His-159 is completely deprotonated between pH 6 and 10. This result is taken as evidence for an imidazolium--thiolate ion-pair interaction between His-159 and Cys-25 wherein neutralization of the charge on the thiolate anion by methylthiolation would be expected to cause a marked decrease in the pK of His-159. A possible catalytic role for the ion pair in the acylation step in papain-catalyzed reactions is proposed wherein attack of a substrate by the imidazolium--thiolate ion pair is accompanied by an increase in the acidity of the imidazolium group that facilitates expulsion of the leaving group of the substrate.

Proton NMR spectroscopy was used to study the ionization behavior of His-159 in a derivative of papain (papain-S-SCH3). In this catalytically inactive derivative of papain, the active-site thiol group of Cys-25 is S-methyl-thiolated so that it cannot form a thiolate anion. The pH dependence of the chemical shift of the C epsilon 1 H resonance of His-159 indicated a pK of 3.45 +/- 0.07 at 45 degrees C in 2H2O with no added ions other than those required for titration. In acetate buffers at an ionic strength of 0.05, the pK increased to 3.87 +/- 0.12. Conversion of papain-S-SCH3 to active papain at pH* 4.17 (at 45 degrees C and an ionic strength of 0.05) caused the position of the C epsilon 1 H resonance to change from a position indicative of partial protonation of His-159 to a position indicative of full protonation, consistent with the existence of an imidazolium-thiolate ion-pair interaction between His-159 and Cys-25 in the active enzyme.

Fluorometric titrations of papain, succinyl-papain, and the corresponding methylthio derivatives of Cys-25 (papain-S-SCH3 and succinyl-papain-S-SCH3) were determined. Removal of the methylthio group from Cys-25 resulted in an increase of approximately 4 pK units in the fluorometrically determined pK value. The correspondence between the ionization behavior as determined by proton NMR and fluorometric titrations indicated that fluorescence titrations reflect the ionization behavior of His-159 in both the active enzyme and the methylthio derivative. The ionic strength dependence of the pK was analyzed in terms of simple electrostatic theory and was shown to be consistent with the charge on the protein. The temperature dependence of the pK values of His-159 indicated an increase in the heat of ionization from about 0 to 8 kcal/mol upon removal of the methylthio blocking group from Cys-25. Measurements of the effect of solvent on the pK's and heats of ionization of simple model compounds indicated that the observed shift in enthalpy of ionization of His-159 upon removal of th methylthio group from Cys-25 is not unreasonable in light of the accompanying perturbation is more than 4 pK units in the pK of His-159. The perturbations in enthalpies and free energies are attributed to formation of an ion pair. The ionization behavior of His-159 in thiol-blocked derivatives of papain is consistent with the involvement of His-159 in the deacylation step in papain catalysis.

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