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.
|Number of pages||6|
|Journal||PROTEIN ENGINEERING DESIGN|
|Publication status||Published - Mar 1997|
- active-site mutants
- site-directed mutagenesis
- MICROBIAL RIBONUCLEASES
- 3-DIMENSIONAL STRUCTURE