206 EMIL L. SMITH, ROBERT L. HILL AND J. R. KIMMEL [II 



explain the effect of pH on ki, since addition or removal of a proton would 

 open the hydrogen bond. Similarly, if there is a preformed thiol ester, it 

 may be postulated that the linkage is opened by changes in pH, but it would 

 be necessary to assume that refolding of the protein could permit regenera- 

 tion of the thiol ester. 



Either hypothesis is attractive since the presence of a 'high-energy' bond 

 would aid in explaining the catalytic activity of the enzyme. Indeed, it is 

 possible that such 'high-energy' bonds are an essential part of the active 

 sites of enzymes and that their formation depends on the specific folding 

 of the protein. It may be noted that the activity of papain is reversibly lost 

 on denaturation with urea. Thus, papain, like other enzymes, depends for 

 its activity on a specific three-dimensional structure despite the fact that a 

 large part of its amino acid sequence is unessential for the maintenance of 

 its activity. 



CONCLUDING REMARKS 



It is evident from the above summary of some of our studies with crystalline 

 papain that only a beginning has been made in understanding how the 

 structure of this protein is related to its function. It will take a long time 

 before the mechanism of action and the specificity of this enzyme can be 

 related to its structure. Nevertheless, it is satisfying that, in our approach 

 to the biological activity of the proteins, we now have the general know- 

 ledge that considerable modification of structure is possible without altera- 

 tion of the activity or specificity of antibodies, protein hormones, enzymes, 

 and viruses. Above all else, the observations made on these substances 

 indicate the general validity of the concept of the active site, namely, that 

 only a part of the structure of a protein molecule is responsible for and 

 involved in its specific interactions. Future progress in the isolation and 

 identification of such active sites or regions of proteins will depend as much 

 on our choice of proteins for study, as on the available tools and methods. 

 The proteolytic enzymes will continue to play a dual role in such studies, 

 since, as this symposium illustrates, everyone must use these enzymes in 

 his working methods with other proteins. At the same time, they are among 

 the more favorable materials for investigation. 



REFERENCES 



1. c. B. ANFINSEN and R. R. REDFIELD, Advances in Protein C/iem., 11, 1 (1956). 



2. J. R. KIMMEL and e. l. smith,/. Biol. Chem., 207, 515 (1954). 



3. J. R. KIMMEL and e. l. smith, Advances in EnzymoL, 19, 267 (1957). 



4. E. L. SMITH, B. J. FiNKLE and A. STOCKELL, Faraday Society Discussions, 20, 



96 (1955). 



5. a. J. FINKLE and e. l. smith,/. Biol. Chem.; in press. 



6. E. L. SMITH, J. R. KIMMEL and D. M. BROWN,/. Biol. Chem., 207, 553 (1954). 



7. E. o. p. THOMPSON, /. 5to/. CA^w., 207, 563 (1954). 



