RESIDUES-36-42 OF LIVER RNASE PL3 CONTRIBUTE TO ITS URIDINE-PREFERRING SUBSTRATE-SPECIFICITY - CLONING OF THE CDNA AND SITE-DIRECTED MUTAGENESIS STUDIES

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative memb ers of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family , RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL 3 is a secreted RNase. Expression of the cDNA in Escherichia coli yiel ded 1.5 mg of purified protein/liter of culture. The recombinant enzym e was indistinguishable from the enzyme isolated from porcine liver ba sed on the following criteria: amino acid analysis, N-terminal amino a cid sequence, molecular weight, specific activity toward yeast RNA, an d kinetic parameters for the hydrolysis of uridylyl(3,5')adenosine and cytidylyl(3,5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compar ed to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most memb ers of the RNase superfamily, strongly prefers uridine over cytidine o n the 5' side of the scissile bond. Replacement, by site-directed muta genesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase i n the catalytic efficiency for cytidine-containing substrates. This su ggests that this region of the molecule contains some of the elements that determine substrate specificity.