196 6. INTERACTIONS OF INHIBITORS WITH ENZYMES 



these amino acids to the active site is obscure. It is possible that histidine 

 is involved in the reaction of DFP with serine and hence occurs at the ac- 

 tive site but in another portion of the polypeptide chain. The group p.K'^'s 

 of the active sites of a variety of enzymes have allowed tentative identifi- 

 cation of histidine as a component. It is probable that histidine is an 

 important constituent of many enzyme active sites since it may function 

 efficiently in acid-base catalysis, due to its piiig being near neutrality. In- 

 direct evidence for the participation of histidine in enzyme catalysis comes 

 from studies on the photo-oxidation with visible light in the presence of 

 methylene blue of the imidazole ring in enzymes, this treatment selectively 

 destroying the histidine residues and producing a progressive fall in activity 

 (Weil and Buchert, 1952; Neely, 1959). The ability of the imidazole ring 

 to accelerate the nonenzymic hydrolysis of phenyl acetates (Bender and 

 Turnquest, 1957; Bruice and Schmir, 1957) and to complex with the pyr- 

 idine nucleotides (Eys, 1958) may be indicative of the role histidine plays 

 in certain enzyme reactions. An excellent summary of determinations of 

 the groups and amino acids in enzyme active centers is in the book by 

 Dixon and Webb (1958, p. 492). 



It is possible that some groups participating in catalysis are present at 

 the active center in especially reactive states. The SH group at the sub- 

 strate site of papain appears to be not an ordinary SH group but one 

 that is thermodynamically at a higher energy level and kinetically more 

 reactive (Smith, 1958). It was postulated that this SH group forms a 

 " high-energy " bond with an adjacent carboxyl group, this being made 

 possible by the folded configuration of the native protein, and that these 

 two groups function as follows in the hydrolysis of peptide bonds: 



enzyme enzyme 



V. J enzyme ^ -J 



S— C Q -) S— C 



ji + H,o S OOC |Jj 



>- I > 



+ R— C ^ + + 



+ ^^* R— COO- 



R— C— NH 

 







The behavior of groups in special states with respect to pH or to reaction 

 with group-specific reagents may deviate from the. predicted on the basis 

 of the same groups on amino acids. 



Another interesting approach, applicable to enzymes that occur in an 

 inactive or zymogen form, should be mentioned at this point. The formation 

 of active trypsin from trypsinogen involves the splitting off of a hexapep- 

 tide val-(asp)4-lys to leave a terminal isoleucine group; thus the breaking 

 of a single peptide bond can result in the appearance of the active site. 



