title: Active site characterization of RNase Rs from Rhizopus stolonifer: involvement of histidine and lysine in catalysis and carboxylate in substrate binding.
abstract: Chemical modification studies on purified RNase Rs revealed the involvement of a single histidine, lysine and carboxylate residue in the catalytic activity of the enzyme. RNA could not protect the enzyme against DEP- and TNBS-mediated inactivation whereas, substrate protection was observed in case of EDAC-mediated inactivation of the enzyme. K(m) and k(cat) values of the partially inactivated enzyme samples suggested that while histidine and lysine are involved in catalysis, carboxylate is involved in substrate binding. Active site nature of RNase Rs suggests that the inability of the enzyme to readily convert 2',3'-cyclic nucleotides to 3'-mononucleotides is probably due to the absence of catalytically active second histidine residue.
authors.affiliation: Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India.
authors.forename: S
authors.initials: S
authors.lastname: Rangarajan
authors.forename: R
authors.initials: R
authors.lastname: Chacko
authors.forename: V
authors.initials: V
authors.lastname: Shankar
mesh_terms: D001665:Binding Sites; D002234:Carbodiimides; D002264:Carboxylic Acids / Q000737:chemistry; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008751:Methylene Blue; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010084:Oxidation-Reduction; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D014302:Trinitrobenzenesulfonic Acid
chemical_list: D002234:Carbodiimides; D002264:Carboxylic Acids; D009712:Nucleotides, Cyclic; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D006639:Histidine; D014302:Trinitrobenzenesulfonic Acid; D004722:Endoribonucleases; C022543:ribonuclease T(2); D008239:Lysine; D008751:Methylene Blue