title: Comparison of the substrate specificity of two potyvirus proteases.
abstract: The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary. tozser@indi.biochem.dote.hu
authors.forename: József
authors.initials: J
authors.lastname: Tözsér
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tropea
authors.forename: Scott
authors.initials: S
authors.lastname: Cherry
authors.forename: Peter
authors.initials: P
authors.lastname: Bagossi
authors.forename: Terry D
authors.initials: TD
authors.lastname: Copeland
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: David S
authors.initials: DS
authors.lastname: Waugh
mesh_terms: D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017800:Potyvirus / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D010450:Endopeptidases; C099488:TEV protease; C059992:nuclear inclusion protein a, mosaic viruses