Evidence for Multiple Reactive Forms of Papain

Abstract

The pH-dependence of the second-order rate constants of acylation and alkylation reactions of the -SH group of papain were determined by using neutral and charged reactants under identical conditions. From these pH rate profiles, in contrast to previous claims, different pK, values were obtained with different groups of reactants. In the case of charged reactants, like chloroacetate (pKa = 3.6) and arginine derivatives (pKa = 4.3), the pK, differences can be attributed to electrostatic effects. However, the fact that a pKa of 3.25 was found with methyl and ethyl bromoacetate, and a pKa of 4.0 was obtained with bromoacetamide and a number of neutral substrates, IS inconsistent with the theories put forth hitherto for the meaning of such pK, values, because they all consider only one reactive enzyme form. The different pKa values obtained here with neutral reactants are explained in terms of various reactive papain forms.The perturbation of pK, by electrostatic effects was examined by reacting simple thiol compounds containing different charges, like 2-mercaptoacetate, 2-mercaptoethylamine, 2-mercaptoethanol and glutathione, with the neutral chloroacetamide and with the negatively charged chloroacetate. Differences in pK, can be interpreted in terms of intramolecular and intermolecular electrostatic interactions.It has been shown by Peller and Alberty [l] that the pK, values calculated from the pH-dependence of k,,,/K,, which equals the second-order rate constant of the reaction between enzyme and substrate [2], are characteristic of the prototropic groups essential for the activity of the free enzyme if ionizing groups on the substrate do not dissociate in the same pH range. This approach has been used for identifying functional groups in enzymes and more frequently misused inasmuch as the assumptions made by Peller and Alberty were blithely disregarded in many instances [3].

Probing cellular protein targets of H2O2 with fluorescein‐conjugated iodoacetamide and antibodies to fluorescein

“…Papain, which contains an essential cysteine with a p K a of 4.0 [12], was subjected to oxidation by 0.2 mM H 2 O 2 at pH 5.0, 6.0, or 7.0, and then to modification by 5‐IAF. Papain treated at each of the three pH values showed negligible reactivity with 5‐IAF, compared with that of the protein not exposed to H 2 O 2 (Fig.…”

Deuterium Isotope Effects on Papain Acylation

“…All papain reactions were performed at 25°C at an ionic strength of 0.09 M in the presence of 1 mM EDTA as described recently in detail [20]. The concentrations of the reactants in the reaction mixture are listed in Table 1.…”

From “fluctuation fit” to “conformational selection”: Evolution, rediscovery, and integration of a concept

“…Instead of a fit induced by the substrate, I would suggest a fluctuating enzyme molecule, one particular form of which is able to bind the substrate and other forms another ligand ” 10. Using the same method of assessing sulfhydryl reactivity, cysteine proteases have provided further experimental evidence for co‐existing molecular conformations in protein‐ligand mixtures 11, 12. H‐isotope exchange experiments 13, 14 maintained this concept until the advent of molecular dynamics simulations and high‐resolution proton NMR measurements on proteins.…”