| Abstract
| - The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordicacharantia), consists of 190 amino acids and is characterized by specific cleavage at the 5‘-side of uridine.Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73,and Arg74, at the α4−α5 loop between α4 and α5 helices for recognition of uracil base by RNase MC1.Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylicacid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect toenzymatic activity. Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutantN71T exhibited 7.0-fold increased Km and 2.3-fold decreased kcat, while the mutant L73A had 14.4-foldincreased Km, although it did retain the kcat value comparable to that of the wild-type. In contrast,replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had littleeffect on the enzymatic activity. This observation is consistent with findings in the crystal structure analysisthat Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1. The role of Asn71 inenzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln,and Asp. Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogenbonding, and the other is to stabilize the conformation of the α4−α5 loop by hydrogen bonding to thepeptide backbone, events which possibly result in an appropriate orientation of the α-helix (α5) containingactive site residues. Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity,as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA andhomopolynucleotides.
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