Identification by site-directed mutagenesis of amino acids in the subsite of bovine pancreatic ribonuclease A |
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Authors: | Tarragona-Fiol, A. Eggelte, H.J. Harbron, S. Sanchez, E. Taylorson, C.J. Ward, J.M. Rabin, B.R. |
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Affiliation: | London Biotechnology Ltd, Department of Biochemistry and Molecular Biology University College London, Gower St., London WC1E 6BT, UK |
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Abstract: | In addition to hydrolysing RNA, bovine pancreatic ribonucleasesplits esters of pyrimidine nucleoside 3'-phosphates, includingdinucleotides. For a series of 3':5'-linked dinucleotides ofgeneral structure CpN, where N is a 5' linked nucleoside, kcalfor the release of N varies enormously with the precise structureof N. Structural studies have been interpreted to indicate thatthe group N interacts with a subsite, B2, on the enzyme thatcomprises Gln69, Asn71 and Glulll. We report studies by site-directedmutagenesis that indicate that Gln69 is not involved in productiveinteractions with any of the dinucleotide substrates and thatAsn71 is an important component of subsite B2 for all dinucleotidesubstrates tested. Glulll appears to be functionally involvedin catalysis for dinucleotide substrates solely when N is guanosine. |
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Keywords: | ribonuclease A/ site-directed mutagenesis/ subsite |
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