204 EMIL L. SMITH, ROBERT L. HILL AND J. R. KIMMEL [11 

 to an important role of an ionized carboxyl group in this reaction. A tenta- 

 tive reaction mechanism for the action of papain has been suggested.^** 

 The main feature of this scheme is that a thiol ester is formed in the enzyme 

 substrate complex. 



Briefly, the following types of observations on papain must be considered. 

 1. The most sensitive known substrates are those which contain the carb- 

 oxyl group of an arginyl or lysyl residue linked to the sensitive bond; i.e., 

 the compounds have a strongly cationic group on the side chain. 2. Papain 

 can hydrolyze synthetic substrates which possess ester^ or thiol ester** bonds, 

 as well as those with peptide or amide bonds. 3. In addition to hydrolytic 

 reactions, papain catalyzes synthetic and transfer reactions. ^-^^ 4. There is 

 one sulfhydryl group which is absolutely essential for the action of papain.^ 



Formation of a thiol ester is consistent with the spatial and chemical 

 specificity of papain which is mainly toward the amino acid residue bearing 

 the carbonyl group of the sensitive peptide bond. The moiety which is dis- 

 placed from or transferred to the carbon of this carbonyl group may be 

 any one of a variety of compounds, e.g., a peptide, an amino acid, ammonia, 

 aniline, hydrazine, hydroxylamine, an alcohol, a thiol, etc.^ 



Postulation of a thiol ester intermediate would appear to be necessary to 

 explain the efiiciency of papain in catalysing transfer reactions, since the 

 replacement reagent R- NHg competes with water or the ions of water for 

 the hydrolytic reaction. The expected free energy change for the scission of 

 the thiol ester bond would indicate that the equihbrium would greatly favor 

 hydrolysis. It may also be emphasized, that as in the case of ficin,*** we 

 have found that the amide and the ethyl ester of benzoyl-L-arginine are 

 split at about the same rate.*^ In fact, the kQ values are almost identical. 

 This suggests that the cleavage step, in the overall reaction, involves a 

 common intermediate, such as the postulated thiol ester. In view of the usual 

 large difference in free energy of activation for hydrolysis of similar esters 

 and amides, the similar Uq values are hard to explain except in terms of an 

 identical intermediate. 



Before concluding, it may be emphasized that there is one major objec- 

 tion to the view that a thiol ester is formed with a free thiol group of the 

 enzyme. It is very likely that the overall free energy of formation of a thiol 

 ester is much greater than that of the susceptible peptide or ester bond. 

 This, in effect, would make the overall reaction scheme energetically im- 

 possible, unless the rate of scission of the thiol ester is sufficient to drive 

 the reaction forward. 



A possible explanation is that a free thiol group does not exist as such 

 in active papain. Instead, there may be present either a thiol group bonded 

 as a high energy hydrogen bond to the titrable carboxylate ion or that a 

 thiol ester, formed by coupling of a carboxyl group and a thiol group, exists 

 preformed in the papain itself. Let us consider the latter possibility first. 

 This hypothesis is attractive insofar as the initial reaction between enzyme 



