CHYMOTRYPSIN AND OTHER PROTEOLYTIC ENZYMES 369 



or nicotinyl), and Eg is a group forming either an amide or ester bond. 

 A typical substrate is benzoyl-L-tyrosinamide: 



NH— CO 

 KO—(. />— CH,— CH 



\\ // Vo-NH 



o 



In addition, the NH — R2 chain may be replaced by H, CI, or OH groups. 

 Only the derivatives of L-amino acids are hydrolyzed. The R^ and Rg 

 groups are important in binding to the enzyme and thus, with the esteratic 

 (paptidatic) site, one may again visualize a three-point attachment. Analogs 

 either devoid of susceptible amide or ester bonds, or having in their 

 place bonds resistent to hydrolysis, are often inhibitory. The R^ group is the 

 most important for binding, as is shown by the strong inhibitory activity 

 of /5-phenylpropionate (hydrocinnamate) (Kaufman and Neurath, 1949). 

 The necessity for at least one aromatic ring in one of the side chains was 

 pointed out by Neurath and Gladner (1951). Their data on the /^-substituted 

 propionates indicate the ring groups to have the following order of inhibitory 

 activity: 



Indole > napthyl > phenyl > 2,4-dinitrophenyl > cyclohexyl 



The distance between the COO" group and the Rj group is also of impor- 

 tance. The inhibitions are summarized in Table 2-19. The weaker binding 

 of cyclohexyl derivatives compared to phenyl compounds (0.5-1 kcal/mole) 

 could be explained by either the smaller polarizability of the cyclohexyl 

 ring or the inability of the cyclohexyl ring to approach the enzyme surface 

 as close as the phenyl ring. The strong binding of the indole compounds 

 was explained on the basis of the enhancement of hydrogen bonding by the 

 ring N. In fact, Neurath and Gladner interpreted the inhibitions by most 

 of the analogs in terms of hydrogen bonding. Even the C00~ may not 

 interact electrostatically with an enzyme cationic group since the binding 

 energies are quite low; indeed, it may serve as a hydrogen acceptor. The 

 equivalent bindings of 



. /y — CH2CH2— COO and (^ /)— O-CH^CH,— OH 



l3-Phenylpropionate 2-Phenoxyethanol 



would be difficult to explain otherwise; however, the latter compound can 

 act as a hydrogen donor in forming a hydrogen bond. These two com- 

 pounds have essentially the same molecular dimensions but the electronic 

 configurations of the terminal groups differ. 



