The Structure and Function of Ribonuclease T1

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Abstract
When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37°, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50° and 1 h at 60°. 3′-GMP and guanosine protected the enzyme significantly from tryptic inactivation. Upon tryptic digestion at 50°, the Lys-Tyr (41–42) and Arg-Val (77–78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. When the trinitrophenylated enzyme, in which the α-amino group of alanine-1 and the ε-amino group of lysine 41 were selectively modified, was treated with trypsin at 37°, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.