Active site characterization of RNase Rs from Rhizopus stolonifer: involvement of histidine and lysine in catalysis and carboxylate in substrate binding

Chemical modification studies on purified RNase Rs revealed the involvement of a single histidine, lysine and carboxylate residue in the catalytic act ivity of the enzyme. RNA could not protect the enzyme against DEP- and TNBS -mediated inactivation whereas, substrate protection was observed in case o f EDAC-mediated inactivation of the enzyme. K-m and k(cat) values of the pa rtially inactivated enzyme samples suggested that while histidine and lysin e 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. (C) 1 999 Elsevier Science B.V. All rights reserved.