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RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations

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JournalPROTEIN ENGINEERING DESIGN
DatePublished - Mar 1997
Issue number3
Volume10
Number of pages6
Pages (from-to)273-278
Original languageEnglish

Abstract

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate, The extracellular ribonuclease from Bacillus intermedius (binase, RNase Ri) shares a common mechanism for RNA hydrolysis with mammalian RNases, Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage, Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GPC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied, The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.

    Research areas

  • active-site mutants, binase, ribonuclease, site-directed mutagenesis, MICROBIAL RIBONUCLEASES, 3-DIMENSIONAL STRUCTURE, BARNASE, RESOLUTION, EXPRESSION, SPECIFICITY, REFINEMENT, INHIBITOR, MECHANISM, SUBSITES

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