11] STRUCTURE AND ACTIVITY OF PAPAIN 205 



and substrate would then involve a transfer reaction with no net energy 

 change to yield an acyl amino acid thiol ester, and this would be followed 

 by a displacement reaction reforming the thiol ester of the enzyme and 

 liberating the acyl amino acid derived from the substrate. Thus, the major 

 part of the enzymic reaction would consist of two transfer reactions involv- 

 ing essentially equivalent thiol ester exchanges resulting in the hydrolysis 

 of the substrate. 



This possibihty is also interesting since it would explain the catalytic 

 efficiency of the enzyme more readily than does the view that an ordinary 

 thiol group is involved. However, we do not know enough, as yet, to decide 

 whether this is more than an interesting possibility. Some of the evidence 

 at hand does not seem to fit this hypothesis too well, but other evidence 

 would fit very nicely indeed. Just two points may be mentioned. As noted 

 above, the activity of papain is related to the amount of sulfhydryl which 

 reacts rapidly in water with /7-chloromercuribenzoate (Fig. 1). The rapidly 

 reacting SH may be in a high energy form which is kinetically more reactive 

 than ordinary sulfhydryl. 



The other finding, which might be cited, is that for four substrates, whose 

 kinetic constants have been measured, the value of ätq is related to the value 

 of k^. This is shown in the double logarithmic plot of Fig. 1 1.^^ If we attach 



LOG k 



I 



Fig. 11. A hypothetical, tentative scheme for the hydrolytic action of papain. (From 

 Stocken and Smith (9).) 



the usual meaning to these constants, the relationship suggests that the 

 more readily ES forms, the more readily it breaks down. Such a view would 

 be in accord with the idea that both reactions, i.e., those involving k^ and âtq, 

 involve chemically similar steps, namely transfer reactions of thiol esters. It 

 may be remarked, in passing, that the relationship illustrated in Fig. 1 1 has 

 also been found for a number of related substrates for carboxypeptidase.*' 

 The alternative hypothesis that the enzyme contains RCOO~ linked to 

 R'SH to form a hydrogen-bonded structure, RCOO" — HSR', would readily 



