56 C. H. W. HIRS, M. HALMANN, J. H. KYCIA 



of inhibitor is present per molecule of enzyme. This makes it likely that 

 the e-amino group of this residue is closely related to the catal}^ic function 

 of the protein. Substitution on lysine may, because of the size of the sub- 

 stituent, locally distort the tertiary structure, or prevent the formation of a 

 critical hydrogen bond required to maintain the configuration of the 

 active site during catalysis. Substitution may also, by removing a potential 

 positive charge, cause a collapse of an electrostatically maintained con- 

 figuration, or prevent electrostatic interaction with the substrate. Finally, 

 the introduced dinitrophenyl group may be efi^ective in preventing bound 

 substrate from interaction with the catalytically essential, bond-breaking 

 amino acids. Further work will permit a narrowing down of the possible 

 alternatives. In the meantime, it is worth recalling that in our earlier 

 work, the speculation was made [i] that the residues between positions 31 

 and 41 might constitute a binding site for anions. A similar idea was recently 

 expressed by Parks [20], who has proposed a model for the tertiary structure 

 and the mechanism of action of ribonuclease. 



Inactivation of ribonuclease by modification of lysine residues has 

 been observed previously. Gundlach et al. [8] have shown that carboxy- 

 methylation of ribonuclease A at pH 8 is accompanied by the inactivation 

 of the enzyme, and that, under these conditions, the reaction is limited 

 to the lysine residues in the protein. Taborsky has phosphorylated ribonu- 

 clease A [21] with imidazole phosphate and has isolated an inactive mono- 

 phospho ribonuclease in which the introduced phospho group is on a 

 lysine residue. Carboxymethylation and phosphorylation on e-amino 

 groups results in a charge reversal, whereas introduction of a dinitrophenyl 

 group makes the lysine residue neutral. If, as seems likely, the same lysine 

 residue is involved in the inactivation reaction with iodoacetate, imidazole 

 phosphate, and fluorodinitrobenzene, it is possible that the groups intro- 

 duced effect inactivation by different mechanisms. 



In conclusion we may briefly list the structural features in ribonuclease 

 A now known to be important in the catalytic action of the protein. 

 Studies on ribonuclease S (for summary, cf. Richards [22]) have demon- 

 strated that the binding of S-peptide (residues 1-20 in the original pro- 

 tein) is essential to the maintenance of activity in ribonuclease S. Carboxy- 

 methylation at pH 5 results in inactivation of ribonuclease A [8] by reaction 

 of the histidine residue at position 119 [23]. Limited pepsin degradation 

 has revealed that aspartic acid residue 121 is required for the maintenance 

 of activity in ribonuclease A [24]. With the implication of the lysine 

 residue at position 41, it is becoming increasingly clear that for the 

 maintenance of the configuration of the catalytic and binding sites of the 

 molecule, the co-operative interaction of many functional groups, located 

 at widely separated points in the primary structural formula, is 

 required. 



