Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity

J Mol Biol. 1996 Apr 19;257(5):992-1007. doi: 10.1006/jmbi.1996.0218.


A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily. Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals. To explore the structural basis of these differences among members in the RNAse family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coli with final yields of 10 to 50 mg per liter of culture after purification. A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity. Cleavage of the recombinant onconase Met-1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity. Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated. Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C). Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity. The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Cell Survival / drug effects*
  • Egg Proteins / antagonists & inhibitors
  • Egg Proteins / chemistry
  • Egg Proteins / metabolism*
  • Egg Proteins / pharmacology
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis
  • Osmolar Concentration
  • Placental Hormones / pharmacology*
  • Pyrrolidonecarboxylic Acid / chemistry
  • Pyrrolidonecarboxylic Acid / metabolism
  • RNA / metabolism
  • Rana pipiens
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Ribonuclease, Pancreatic / antagonists & inhibitors
  • Ribonuclease, Pancreatic / chemistry
  • Ribonuclease, Pancreatic / metabolism*
  • Ribonuclease, Pancreatic / pharmacology
  • Ribonucleases / antagonists & inhibitors
  • Ribonucleases / chemistry
  • Ribonucleases / metabolism*
  • Ribonucleases / pharmacology
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Tumor Cells, Cultured


  • Egg Proteins
  • Enzyme Inhibitors
  • Placental Hormones
  • Recombinant Fusion Proteins
  • placental ribonuclease inhibitor
  • RNA
  • Ribonucleases
  • Ribonuclease, Pancreatic
  • Pyrrolidonecarboxylic Acid
  • ranpirnase