The Structural Roles of Amino Acid Residues Near the Carboxyl Terminus of Bovine Pancreatic Ribonuclease A

Abstract The sequence at the COOH terminus of bovine pancreatic ribonuclease A is known to be -Pro-Val-His-Phe-Asp-Ala-Ser-Val. When the last 4 residues are removed by peptic hydrolysis, the resulting des-(121-124)-RNase, which was earlier thought to be inactive, is now shown to possess about 0.5% of the activity of the native enzyme toward cyclic 2',3'-cytidylic acid. Several types of experiments show that this activity is intrinsic. The highly purified derivative binds the inhibitor 2'-cytidylic acid at pH 5.5 with a dissociation constant of 40 µm compared with 3 µm for native RNase. When the activity of the derivative is measured toward cyclic cytidylic acid, Km is 0.5 mm and the turnover number is 2 x 10-3 sec-1; these values represent a Km that is twice that of RNase but an activity that is 0.005 that of the initial enzyme. These findings are consistent with results on the alkylation of residues that are considered part of the active site of RNase; in the des-(121-124)-derivative the alkylation of histidine-12 and lysine-41 is still strongly facilitated but the susceptibility of histidine-119 is diminished. An increase in the pK of histidine-119 which could account for the alkylation results could also contribute to the diminution of the catalytic activity. The relatively strong binding of inhibitor by des-(121-124)-RNase suggests that serine-123 can have only a minor role in binding substrate. However, when phenylalanine-120 is removed from the des-(121-124)-derivative by controlled hydrolysis with carboxypeptidase A at pH 5 and 45°, the activity, the ability to bind inhibitor, and the susceptibilities of the histidine residues to alkylation are lost. The transition temperature of the des-(120-124)-derivative is lowered to 34° from 61° for RNase A. The phenylalanine residue, which in the three-dimensional structure fits into a hydrophobic pocket in the molecule, thus has an important role in maintenance of the native conformation of the enzyme. Histidine-119 as well as phenylalanine-120 can be removed by hydrolysis of the des-(121-124)-derivative by carboxypeptidase A at pH 7.6. The molecule with the 6 residues removed has a transition temperature of 32° and a structure highly susceptible to proteolysis by trypsin.