CHYMOTRYPSIN AND OTHER PROTEOLYTIC ENZYMES 373 



tryptaraine indicates also a more favorable field for positive than negative 

 ionic groups, but the positive charge is not important since acetyltrypt- 

 amine is bound even more readily. 



Foster and Niemann (1955 a) determined the values of K^ for several 

 inhibitors at pH 6.9 and 7.9. The acetyltryptophanates and indolepropio- 

 nate are bound 1.0-1.4 kcal/mole more tightly at pH 6.9 than at pH 7.9, 

 whereas the binding of various amides is not significantly affected. This is 

 interpreted as pointing to the development of a negative charge in the vicin- 

 ity of the active site as the pH rises from 6.9 to 7.9; this charge would repel 

 the negatively charged tryptophanates and other carboxylates. It is pos- 

 sible that the lack of effect of pH on amide binding is due to the simulta- 

 neous deprotonation of the CONH3+ with rise in pH (p^^ = 7.5). The con- 

 cept of a negative charge on or near the active center was first postulated 

 by Neurath and Schwert (1960) on the basis of the suppressing effect of a 

 carboxylate group on the hydrolysis of an adjacent peptide bond. Whether 

 this enzyme negative group participates in the hydrolysis along the lines 

 suggested by Steam (1935) and Vaslow and Doherty (1953) is not certain. 



The nitrogen analogs of substrates are usually not hydrolyzed and can 

 act as inhibitors (Kurtz and Niemann, 1961). In these compounds an a- 

 methine group is replaced by a N atom, or an or-methylene group by an 

 NH group. Thus ethyl l-acetyl-2-benzylcarbazate is an analog of ethyl 



^COO— Et 



CH2— N 



NH- COCH3 



acetyl-L-phenylalaninate and is an inhibitor, with K^ = 20 raM. Also 

 9)— CH2CH2— COO— CH3 and (7^— NH— CH2— COO— CH3 are substrates of 

 chymotrypsin whereas cp — CHg — NH — COO — C2H5 is an inhibitor [K^ = 

 6 m.M). It is suggested that an a-N= or a-NH — group leads to a restriction 

 of the rotation around the bond joining it to the CO group compared to 

 that of a C — C bond, this constraint leading to loss of substrate activity or 

 weakening of inhibitor binding. 



Wallace et al. (1963) published the results of their studies of the interac- 

 tions of 136 compounds with chymotrypsin, and it is seen that several 

 substances not directly related to amino acids are fairly potent inhibitors; 

 such are quinoline and the methylquinolines, hydroxyquinolines, and ami- 

 noquinolines, various acridines, a-naphthol, and the naphthylamines, all 

 with K^ values less than 1 mM . They summarized their conclusions with re- 

 spect to structure and inhibitory activity in ten postulates, from which the 

 following comments are extracted. Aromatic compounds are more effective 

 than the corresponding saturated derivatives, and monosubstituted benzene 

 derivatives with polarizable uncharged groups are more inhibitory than the 



