On the Mode of Activation of the Catalytically Essential Sulfhydryl Group of Papain

Abstract

The reaction of the thiol group of papain with chloroacetamide, iodoacetamide, D-and L-2-bromopropionamides was studied. I n the acidic pH-range the reaction rate is higher than expected for an ordinary SH-group and shows a double-sigmoid pH-rate profile. I n addition to the already described pK, of 8.5, we found a pK, of 4.0. This indicates that in the pH-range where the enzyme is catalytically active the thiol group of Cys-25 interacts with some amino acid side chain, presumably with the imidazole group of the neighboring His-159. I n the light of the X-ray diffraction studies (Drenth et al., 1971), it is probable that the anomalous PKa of His-I59 is due to its interaction with Trp-177.The comparison of the pH-rate profiles of D-and L-2-bromopropionamide reactions indicates that there is no sifgnificant alteration in the geometry of the active site around pH 4, whereas a t slightly alkaline p H structural changes can be observed.2H,O has no effect on the rate constants of the alkylation reaction. This rules out the possibility of general base catalysis by the imidazole group of His-159.The experimental data indicate that the dissociated thiol group forms an ion pair with the protonated imidazole group. On the basis of the formation of thiolate-imidazolium ion pair, the mechanism of action of papain is reconsidered. It is concluded that the acylation and deacylation steps are not symmetrical processes, and they are one-encounter type reactions. The interaction between His-I59 and Trp-177 may be important in the catalysis.The pHdependence of the reaction of papain with chloroacetamide [I] and 2-iodopropionamide [2] is similar to that of an ordinary SH-compound. The second-order rate constants with the enzyme are about 10 times higher [I, 21. These investigations were restricted to the alkaline pH-range where the dissociated SH-group of papain is a very good nucleophile in a simple alkylation reaction. However, if the SH-group is activated by some amino acid side chain, this is to be expected rather in the acidic pH-range where papain shows high enzymatic activity. The formation of acyl-papain displays a bell-shaped pH-rate profile with pK, -4 and pK, B 8.5 [3]. The present study is focused on the acidic pH-range. It is found that with decreasing pH the rate of alkylation of the SH-group becomes higher by several orders of magnitude than that of a normal SH-group. This allows us to draw conclusions about the nature of the tjhiol group not only in the alkylation but also in the enzymatic catalysis .Abbreviation. Nbs,, 5,5'-dithio-bis(2-nitrobenzoic acid) (formerly abbreviated DTNB).Enzyme. Papain (EC 3.4.4.10). MATERIALS AND METHODSChemicals 5,5'-Dithio-bis(2-nitrobenzoic acid) (Nbs,) was purchased from Fluka AG and carbobenzyloxyglycine nitrophenyl ester from Calbiochem Co. Iodoacetamide and chloroacetamide were recrystallized from carbon tetrachloride and water, respectively ; D-and L-2-bromopropionamide were synthesized by Dr E. Moravscik. The enrichment of 2H20 was greater than 99O/,. Enzyme 0.3 ml of twice-cr...

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