Further Studies on the Alkylation of the Histidine Residues at the Active Site of Pancreatic Ribonuclease

The chemistry of the active site of bovine pancreatic ribonuclease has been further examined by study of the reaction of iodoacetic acid with histidine residues 12 and 119 in derivatives of the protein. the native enzyme, the stereospecific alkylation of the two imidazole rings is a mutually exclusive, electrostatically oriented reaction leading to a 7:1 ratio of substitution at residues 119 and 12. When inactive des-(121-124)-ribonuclease, prepared by limited peptic hydrolysis according to the method of Anfinsen, is exposed to iodoacetate at pH 5.5, alkylation at histidine-119 is nearly abolished but the reaction at histidine-12 is accelerated. If, as has been assumed for the native enzyme, 1 protonated histidine residue orients the reagent for the alkylation of the unprotonated form of the other, this result with the derivative is interpretable in terms of an increase in the pK of the imidazole ring of histidine-119 accompanying the removal of -Asp-Ala-Ser-Val from the carboxyl end of the chain. In turn, this change in pK would alter the precise interaction between the two imidazole rings which are essential for catalytic action; such a shift could account for the inactivity of the des-(121-124)-derivative. support this interpretation of the alkylation of des-(121-124)-ribonuclease, further information on the alkylation reaction itself was sought. The influence of neighboring groups on the course of the alkylation of ribonuclease has been studied with two derivatives in which lysine residues have been modified. When the positive charge on lysine-7 is neutralized by acetylation of the S-peptide, by the procedure of Richards and Vithayathil, the resulting active Nα, ε-diAcLys-1-,Nε-Lys(Ac)-7-ribonuclease S is alkylated by iodoacetate mainly on histidine-119, as in the unaltered enzyme. The alkylation also proceeds similarly with the inactive ε-Lys(DNP)-41-ribonuclease. These results eliminate the possibility that the positive charges on the ε-NH2 groups of lysine-7 and lysine-41 have a role in the orientation of ICH2COO-.