title: A ribonuclease A variant with low catalytic activity but high cytotoxicity.
abstract: Onconase, a homolog of ribonuclease A (RNase A) with low ribonucleolytic activity, is cytotoxic and has efficacy as a cancer chemotherapeutic. Here variants of RNase A were used to probe the interplay between ribonucleolytic activity and evasion of the cytosolic ribonuclease inhibitor protein (RI) in the cytotoxicity of ribonucleases. K41R/G88R RNase A is a less active catalyst than G88R RNase A but, surprisingly, is more cytotoxic. Like Onconase, the K41R/G88R variant has a low affinity for RI, which apparently compensates for its low ribonucleolytic activity. In contrast, K41A/G88R RNase A, which has the same affinity for RI as does the K41R/G88R variant, is not cytotoxic. The nontoxic K41A/G88R variant is a much less active catalyst than is the toxic K41R/G88R variant. These data indicate that maintaining sufficient ribonucleolytic activity in the presence of RI is a requirement for a homolog or variant of RNase A to be cytotoxic. This principle can guide the design of new chemotherapeutics based on homologs and variants of RNase A.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: L E
authors.initials: LE
authors.lastname: Bretscher
authors.forename: R L
authors.initials: RL
authors.lastname: Abel
authors.forename: R T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000633:toxicity*; D002455:Cell Division / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004527:Egg Proteins / Q000378:metabolism / Q000633:toxicity; D004795:Enzyme Stability; D014644:Genetic Variation; D006801:Humans; D020014:K562 Cells; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D012260:Ribonucleases / Q000378:metabolism / Q000633:toxicity; D013936:Thymidine / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D004527:Egg Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D013936:Thymidine; C058703:ranpirnase