Altering the Residues of Ribonuclease A

The cDNA that codes for cow pancreatic ribonuclease A has been cloned and expressed in the yeast S. cerevisiae. Site-directed mutagenesis of conserved residues Lys41 and ASP121 has been used to probe catalysis by the enzyme. In addition, mutation of Lysl to cysteine has allowed for the covalent attachment or a DNA oligonucleotide that confers sequence specificity on ribonuclease A. Finally, a nutritional selection and an activity screen have been developed to distinguish yeast colonies that secrete active ribonuclease A. The high ribonuclease activity in the pancreas of ruminants has led to the detailed characterization of ribonuclease A (RNase A; EC 3.1.27.5), the predominant ribonuclease in cow pancreas. RNase A has been a widely used subject for protein folding studies; and its structure, its protein chemistry, and the mechanism and energetics of its catalysis are all well-defined (RICHARDS and WYCKOFF, 1971; KARPEISKY and YAKOVLEV. 1981; BLACKBURN and MOORE. 1982; BEINTEMA. 1987). Although RNase A may be better characterized than any other enzyme, it has only recently been subjected to study by molecular biology (STACKHOUSE et al.. 1990). Two obstacles have been encountered. First. the Figure 1. Mechanism of the reaction catalyzed by ribonuclease A. In the transphosphorylation step. His12 is a general base and Hisl19 is a general acid. In the hydrolysis step, HiS1l9 is a general base and His12 is a general acid. slow