10] ACTIVATION OF TRYPSINOGEN 173 



(c) Interaction between the aspartyl carboxyl groups, electrostatically, with 

 positively charged groups, or by hydrogen bonding with groups elsewhere 

 along the chain, provides a loop which is destroyed when the lysyl-isoleucine 

 bond is broken by trypsin, allowing the newly-formed molecule to coil up 

 and to form the active center of trypsin, (d) Two of the six disulfide bridges 

 of trypsinogen are in the region of the molecule which includes the catalytic 

 site. This follows from studies by Dr G. H. Dixon in our laboratory on the 

 tryptic degradation of oxidized diisopropylphosphoryl (DlP)-trypsin, which 

 have shown that the largest DIP-peptide contained five cysteic acid residues 

 among a total of 55 amino-acid residues, (e) The active center, dehneated 

 here by the dotted line, includes besides the catalytic site, the grouping con- 

 ferring side-chain specificity to the enzyme, as denoted by X. (/) The cata- 

 lytic site involves a histidine and a serine residue. These two groupings are 

 separated from each other in trypsinogen, but upon activation, come suffi- 

 ciently close to become hydrogen-bonded to one another.^ -^ This conclu- 

 sion is based primarily on kinetic studies of the reaction of trypsin and 

 chymotrypsin with acylating agents, such as /7-nitrophenyl acetate (reviewed 

 in ref. 9). 



An essential feature of this scheme is that the splitting of the lysyl-isoleucine 

 bond removes a structural impediment, which allows the A^-terminal sequence 

 of the newly-formed molecule to coil up, thereby bringing the histidine and 

 serine residues into juxtaposition. According to this picture, cleavage of a 

 bond to the left of the lysine residue might not cause activation, as electro- 

 static interaction or hydrogen bonding of the lysine side chain would still 

 provide a certain element of structural rigidity and prevent formation of 

 the helix. On the other hand, the splitting of any bond to the right of the 

 lysine side chain would produce the same result as the splitting of the lysyl- 

 isoleucine bond. According to this interpretation, the structural contribution 

 of two or more hydrogen bonds, or salt linkages, is an important factor in 

 the conversion of the zymogen to the active enzyme, and were it possible 

 to rupture these bonds specifically, activation of trypsinogen might ensue. 

 This has not proved possible by the simple expedient of denaturation of 

 trypsinogen by urea ; in fact, whereas trypsin is still active in 3m urea, tryp- 

 sinogen can no longer be activated under these conditions, even by the 

 addition of trypsin, presumably because other structurally important hydro- 

 gen bonds are dissolved at the same time. 



The highly specific action of trypsin in hydrolysing the lysyl-isoleucine 

 bond may, therefore, be viewed as both a case of limited proteolysis and of 

 limited hydrogen-bond rupture. The question as to whether the hexapeptide 

 is actually liberated during this process or whether it remains attached and 

 is only freed after addition of trichloroacetic acid has not yet been answered 

 in an unequivocal way, but all evidence thus far available suggests strongly 

 that it is liberated as the lysyl-isoleucine bond is broken. 



Details of the present results will be pubhshed elsewhere.^'' 



