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1. The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH-range the -SH group ofthe enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in aHaO it is concluded that a mercaptideimidazolium ion-pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH.2. Iodoacetamide reacts with the -SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo-, chloro-, and fluoroacetamide. This might be due to the less polar environment around the -SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo-and chloroacetamide in the presence of dioxane.3. By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be established above pH z 9, whereas the overall protein structure is stable up to pH zz 11. D-2-Bromo-n-butyramide reacts with thiolsubtilisin about 60 times as fast as D-2-bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala-152 and Thr-220 of thiolsubtilisin may form van der Waals interactions with D-2-bromon-butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D-2-bromopropionamide and thiolsubtilisin.Thiolsubtilisin, an -SH analog of the serine protease subtilbin, can be produced by the replacement of the reactive serine residue at the active site by a cysteine residue [l]. Thiolsubtilisin can be regarded as a model of thiol proteases, such as papain, inasmuch as both thiolsubtilisin [2] and papain [3] have an imidazole ring in the immediate vicinity of the thiol group, as clearly indicated by X-ray diffraction studies. However, thiolsubtilisin is not a protease, it can only catalyze the hydrolysis of active esters [a].In a recent paper we have pointed out that the catalytic action of papain depends on the formation of a mercaptide-imidazolium ion-pair at the active site [5]. On the basis of acylation of thiolsubtilisin, we have also suggested that the reactivity of the thiol group is highly affected by an interaction with the The detailed study of alkylation of Carlsberg thiolsubtilisin is presented in this paper. Unlike acylation, alkylation is a simple nucleophilic displacement reaction, not complicated by the formation of an intermediate, therefore the study of alkylation, rather than of acylation, offers more conclusive evidence for the chemical behavior of the thiol group. Carlsberg thiolsubtilisin was preferred to the Novo enzyme because of its higher stability. We have found that around neutral pH values there is indeed an interaction between the thiol and imi...

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].