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

 sight, might be responsible are an a- or e-ammonium groups, an imida- 

 zoHum, a phenolic or a sulfhydryl group. It is very unlikely that the strongly 

 cationic substrate, benzoylargininamide, could interact with a cationic site 

 on the enzyme. Moreover, the calculated pK' values are much too low for 

 an e-ammonium or a phenolic group, and too high for an imidazolium 

 group. Papain possesses only one a-amino group, but, as already em- 

 phasized, much of the amino-terminal sequence is unessential for the activity 

 of the enzyme. These considerations suggest that the a-amino group does 

 not participate in determining the value of ki. 



In effect, the only remaining group to be considered is the sulfhydryl 

 group and it is already known that such a group is essential for papain 

 activity. It may be noted that the pK' values are in the range recently found 

 for sulfhydryl groups, ^^ but the calculated heat of ionization appears to be 

 rather high. However, these data are rather limited in the critical regions 

 where pH has a marked effect on ki, and the precision is rather low. In 

 subsequent studies, the effect of pH on the hydrolysis of other substrates 

 has been determined and, now knowing the important range to be investi- 

 gated, more complete data have been obtained. 



100 



80- 



^ 60 



°^ 40 — 



20 



3 4 5 6 7 8 9 



pH 



Fig 9. Effect of pH on kx for the hydrolysis of carbobenzoxy-L-histidinamide with the 

 maximum for each temperature set equal to 100. (From Smith, Chavre and Parker (42).) 



Fig. 9 shows the effect of pH on calculated values of k^ for the hydrolysis 

 of carbobenzoxy-L-histidinamide.*^ The data have been fitted by drawing 

 theoretical titration curves for a single titratable group at both sides of the 

 curve. On the acid side of the curve, there is little effect of temperature and 

 the apparent pK' at 38° is near 4-3. On the alkaline side, there is a shift of 

 pK' with temperature and the calculated AW, the heat of ionization, is about 

 4-4 kcal, per mole at 0°. Both the pK' value and the value of /I H are near 



