title: RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations.
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 Bi) 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.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: A L
authors.initials: AL
authors.lastname: Okorokov
authors.forename: K I
authors.initials: KI
authors.lastname: Panov
authors.forename: W A
authors.initials: WA
authors.lastname: Offen
authors.forename: V G
authors.initials: VG
authors.lastname: Mukhortov
authors.forename: A A
authors.initials: AA
authors.lastname: Antson
authors.lastname: Karpeisky MYa
authors.forename: A J
authors.initials: AJ
authors.lastname: Wilkinson
authors.forename: G G
authors.initials: GG
authors.lastname: Dodson
mesh_terms: D001216:Asparagine / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology; D002384:Catalysis; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D012313:RNA / Q000378:metabolism*; D013912:Threonine / Q000235:genetics*
chemical_list: D013912:Threonine; D012313:RNA; D001216:Asparagine; D004722:Endoribonucleases; C022543:ribonuclease T(2)