11] STRUCTURE AND ACTIVITY OF PAPAIN 203 



those expected for a sulfhydryl group. '*^ Studies at 38° of the effect of pH 

 on ki for the hydrolysis of benzoylglycinamide*^ and of benzoyl-L-arginine 

 ethyl ester^'' yield curves with the same shape as those shown for carbo- 

 benzoxy-L-histidinamide. 



The effect of pH on ki for another type of substrate, carbobenzoxygly- 

 cylglycine, is shown in Fig. 10.^"^ On the alkaline side, the curve has been 



Fig. 10. Effect of pH on ki for hydrolysis of carbobenzoxyglycylglycine at 38°. (From 

 Smith, Chavre and Parker (42).) 



fitted by a theoretical titration curve with a pK' near 8-0 which is close to 

 that found for other substrates at 38°. However, there is a plateau near pH 6 

 and ki rises sharply at more acid pH values. It should be emphasized that 

 at pH 6, the absolute value for ki for the scission of carbobenzoxyglycyl- 

 glycine is only about 0-1 per cent of that for benzoylargininamide. At pH 6, 

 both the enzyme and carbobenzoxyglycylglycine would have carboxyl groups 

 in ionized form and electrostatic repulsion may be expected. The increasing 

 values of ki below pH 5-5 would appear to be due to repression of ioniza- 

 tion of one or both carboxyl groups, thus decreasing the degree of repul- 

 sion between enzyme and substrate and, in effect, promoting an increase 

 in the value of ki. 



The kinetic data which have been cited, point to the participation of the 

 essential sulfhydryl group in the ki step. Moreover, since the optimal region 

 of ki is near pH 6, it is evident that the active form of this group is RSH 

 and not RS~. In addition, evidence has been obtained that an ionized carb- 

 oxyl group is involved in the ki step. Inasmuch as the kinetic studies have 

 been performed with rather small substrates, it is likely that the carboxyl 

 group must be close to the sulfhydryl group. 



A TENTATIVE REACTION MECHANISM 



Thus, the evidence from kinetic and other studies indicates that the essential 

 sulfhydryl group participates in the enzyme-substrate interaction and points 



