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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 pKa 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 pKa 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 pKa 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 pKa 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 chioroacetate. Differences in pKa can be interpreted in terms of intramolecular and intermolecular electrostatic interactions. [ABSTRACT FROM AUTHOR]

The reactive mercaptide-imidazolium ion-pair at the active site of papain and thiolsubtilisin was alkylated with negatively charged reactants. The reactivities of the two thiolenzymes differ considerably. The iodoacetate reaction is faster by more than 1000 times with papain than with thiolsubtilisin. On the other hand, towards 3-iodopropionate thiolsubtilisin is more reactive than papain by about a factor of 7. These findings, which are interpreted in terms of the different geometries of the two ion-pairs, offer an explanation of the basic difference between the catalytic abilities of papain and thiolsubtilisin. [ABSTRACT FROM AUTHOR]

A new approach is proposed for probing the polarity of the immediate environment of - SH groups in thiolenzymes, based on the alkylation of the - SH group with methyl iodide, a relatively small and non-polar molecule. Rate and activation parameters (ΔH*, ΔS*) for the reaction of the enzyme are compared to those of glutathione, a simple - SH compound alkylated in aqueous medium. The enzyme and model compound are also reacted with iodoacetamide, a polar counterpart of the non-polar methyl iodide. The above method was applied to thiolsubtilisin, an artificial thiolenzyme. 1. The ratio of the rates of alkylation of thiolsubtilisin and glutathione is about 20 times as high with methyl iodide as with iodoacetamide. 2. ΔH* and ΔS* for enzyme alkylation, as compared to those for glutathione, are remarkably lower with methyl iodide whereas they are slightly higher with iodoacetamide. 3 ΔH* and ΔS* for alkylation of thiolsubtilisin with methyl iodide are similar to those found with glutathione in 40% dioxane/water mixture. 4. The activation enthalpy and entropy values for the reaction of thiolsubtihsin with D-2 bromo-n-valeramide are lower than those for glutathione reaction. Consequently, in this respect, D-2-bromo-n-valeramide is similar to methyl iodide rather than to iodoacetamide. It is concluded that the - SH group of thiolsubtilisin is located in an environment less polar than water. The concentration of methyl iodide in this non-polar layer is higher than in the bulk solution, which results in an enhanced reaction rate. [ABSTRACT FROM AUTHOR]

The effect of the microenvironment on the reactivity of the essential - SH group of papain was studied by alkylation with methyl iodide and with the more polar iodoacetamide. Rate and activation parameters for these reactions were determined with two form of the - SH group: the free mercaptide ion at pH 10.0, and the mercaptide-imidazolium ion-pair at pH 5.5. The ion-pair of papain reacts with methyl iodide at a rate 1470 times less than that of thiolsubtilisin, This suprising difference between the reactivities of the two enzymes suggests that in contrast to thiolsubtilisin, where a non-polar environment enhances the rate, in the case of papain a more polar environment somewhat inhibits the reaction with the non-polar methyl iodide. The positive activation entropy for the papain reaction may indicate an bordered structure of bound water around the sulfur atom. The high rate and the low activation entropy (organized transition state) of the reaction of papain with iodoacetamide can be explained in terms of hydrogen-bond formation between the enzyme and the amide group of the alkylating agent. [ABSTRACT FROM AUTHOR]

The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH-range the —SH group of the 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 ²H2O it is concluded that a mercaptideimidazolium ion-pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH. 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. By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be establisbed above pH &assymp; 9, whereas the overall protein structure is stable up to pH &assymp; 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-bromo-n-butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D-2-bromopropionamide and thiolsubtilisin. [ABSTRACT FROM AUTHOR]