Open AccessOn the Mode of Activation of the Catalytically Essential Sulfhydryl Group of Papain
Author(s) -
Polgár László
Publication year - 1973
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1973.tb02660.x
Subject(s) - chemistry , papain , protonation , thiol , imidazole , iodoacetamide , reaction rate constant , acylation , alkylation , medicinal chemistry , stereochemistry , catalysis , active site , cysteine , ion , organic chemistry , enzyme , kinetics , quantum mechanics , physics
The reaction of the thiol group of papain with chloroacetamide, iodoacetamide, d ‐ and l ‐2‐bromopropionamides was studied. In the acidic pH‐range the reaction rate is higher than expected for an ordinary SH‐group and shows a double‐sigmoid pH‐rate profile. In addition to the already described p K a of 8.5, we found a p K a 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. In the light of the X‐ray diffraction studies (Drenth et al. , 1971), it is probable that the anomalous P K a of His‐159 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 at slightly alkaline pH structural changes can be observed. 2 H 2 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‐159 and Trp‐177 may be important in the catalysis.