CHYMOTRYPSIN AND OTHER PROTEOLYTIC ENZYMES 375 



The hydrolysis of a-benzoyl-L-argininamide by papain is inhibited com- 

 petitively by carbobenzoxy-L-glutamate (Kimmel and Smith, 1954; Stockell 

 and Smith, 1957). The inhibition is very sensitive to pH between 3.9 and 

 5, the inhibitory activity almost disappearing at the upper end of this range 

 (see Fig. 1-14-6). The y-carboxyl group has a pTiT,, of 4.4 so it is possible 

 that the active form is un-ionized: 



coo" 



/ 

 HOOC— CH2CH2— CH /^^ 



Vh^— COO-CH2 — (\ /) 



It is rather surprising that the ionization of this group should be so im- 

 portant in the binding, especially as it is at some distance from the other 

 probable binding groups, and the enzyme as a whole is quite positively 

 charged at these pH's (isoelectric point near 8.75). Perhaps a hydrogen- 

 bonding function must be attributed to the COOJI group instead. Benzoyl- 

 L-arginine {K^ = 60 mM) and benzoyl-L-ar'gininamide (iiC, = 54 mM) 

 also inhibit the hydrolysis of benzoyl-L-arginine ethyl ester (K,,, =23 mM) 

 by ficin at pH 5.5 (Bernhard and Gutfreund, 1956). 



Beef spleen cathepsin C is competitively inhibited by various amides, 

 esters, and dipeptides when glycyl-L-tyrosinamide is the substrate (Fruton 

 and Mycek, 1956). L-Phenylalanine amides and esters are good inhibitors 

 (as shown in the accompanying tabulation) but L-phenylalanine, acetyl-L- 



phenylalanine, and glycyl-DL-phenylalanine are inactive. ^-Acetylation or 

 a free C00~ group seems to prevent binding. The interaction between the 

 inhibitors and the enzyme may involve several groups with possible hydro- 

 gen bonding of the carbonyl group in a manner similar to that proposed for 

 chymotrypsin. Decarboxylation and A^-acylation of amino acids can lead 

 to inhibitors, as in the competitive inhibition by tosylagmatine of thrombin 

 (Ki = 13.2 mM) and trypsin {K, = 3.45 mM) (Lorand and Rule, 1961). 

 This inhibitor markedly slows clotting in a thrombin-fibrinogen system and 

 in whole plasma. 



