Degradation of double-stranded RNA by human pancreatic ribonuclease: crucial role of noncatalytic basic amino acid residues.
Under physiological salt conditions double-stranded (ds) RNA is resistant to the action of most mammalian extracellular ribonucleases (RNases). However, some pancreatic-type RNases are able to degrade dsRNA under conditions in which the activity of bovine RNase A, the prototype of the RNase superfamily, is essentially undetectable. Human pancreatic ribonuclease (HP-RNase) is the most powerful enzyme to degrade dsRNA within the tetrapod RNase superfamily, being 500-fold more active than the orthologous bovine enzyme on this substrate. HP-RNase has basic amino acids at positions where RNase A shows instead neutral residues. We found by modeling that some of these basic charges are located on the periphery of the substrate binding site. To verify the role of these residues in the cleavage of dsRNA, we prepared four variants of HP-RNase: R4A, G38D, K102A, and the triple mutant R4A/G38D/K102A. The overall structure and active site conformation of the variants were not significantly affected by the amino acid substitutions, as deduced from CD spectra and activity on single-stranded RNA substrates. The kinetic parameters of the mutants with double-helical poly(A).poly(U) as a substrate were determined, as well as their helix-destabilizing action on a synthetic DNA substrate. The results obtained indicate that the potent activity of HP-RNase on dsRNA is related to the presence of noncatalytic basic residues which cooperatively contribute to the binding and destabilization of the double-helical RNA molecule. These data and the wide distribution of the enzyme in different organs and body fluids suggest that HP-RNase has evolved to perform both digestive and nondigestive physiological functions.
Sorrentino
Salvatore
S
Department of Biological Chemistry, University Federico II of Naples, Naples, Italy. ssorrent@unina.it
Naddeo
Mariarosaria
M
Russo
Aniello
A
D'Alessio
Giuseppe
G
D019943:Amino Acid Substitution; D024361:Amino Acids, Basic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011067:Poly dA-dT / Q000737:chemistry / Q000378:metabolism; D011131:Polyribonucleotides / Q000737:chemistry / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D055672:Static Electricity; D013223:Statistics as Topic; D013379:Substrate Specificity
D024361:Amino Acids, Basic; D011131:Polyribonucleotides; D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D012367:RNA, Viral; D011994:Recombinant Proteins; D011067:Poly dA-dT; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic