IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS OF AMINO-ACIDS IN THE B2 SUBSITE OF BOVINE PANCREATIC RIBONUCLEASE-A

In addition to hydrolysing RNA, bovine pancreatic ribonuclease splits esters of pyrimidine nucleoside 3'-phosphates, including dinucleotides. For a series of 3':5'-linked dinucleotides of general structure CpN, where N is a 5' linked nucleoside, k(cat) for the release of N varies enormously with the precise structure of N. Structural studies have been interpreted to indicate that the group N interacts with a subsite, B2, on the enzyme that comprises Gln69, Asn71 and Glu111. We report studies by site-directed mutagenesis that indicate that Gln69 is not involved in productive interactions with any of the dinucleotide substrates and that Asn71 is an important component of subsite B2 for all dinucleotide substrates tested. Glu111 appears to be functionally involved in catalysis for dinucleotide substrates solely when N is guanosine.