title: Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.
abstract: Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.
authors.affiliation: Department of Microbiology, School of Medicine, State University of New York, Stony Brook 11794.
authors.forename: T
authors.initials: T
authors.lastname: Hämmerle
authors.forename: C U
authors.initials: CU
authors.lastname: Hellen
authors.forename: E
authors.initials: E
authors.lastname: Wimmer
mesh_terms: D000595:Amino Acid Sequence; D001345:Autoradiography; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology*; D014158:Transcription, Genetic
chemical_list: D015345:Oligonucleotide Probes; D010450:Endopeptidases