title: Yellow fever virus NS2B/NS3 protease: hydrolytic properties and substrate specificity.
abstract: Here we report the hydrolytic behavior of recombinant YFV NS2B/NS3 protease against FRET substrates mimicking the prime and non-prime region of the natural polyprotein cleavage sites. While the P2-P'1 motif is the main factor associated with the catalytic efficiency of Dengue (DV) and West Nile Virus (WNV) protease, we show that the k(cat)/K(m) of YFV NS2B/NS3 varied by more than two orders of magnitude, despite the presence of the same motif in all natural substrates. The catalytic significance of this homogeneity - a unique feature among worldwide prominent flavivirus - was kinetically analyzed using FRET peptides containing all possible combinations of two and three basic amino acids in tandem, and Arg and Lys residues produced distinct effects on k(cat)/K(m). The parallel of our data with those obtained in vivo by Chambers et al. (1991) restrains the idea that these sites co-evolved with the NS2B/NS3 protease to promote highly efficient hydrolysis and supports the notion that secondary substrate interaction distant from cleavage sites are the main factor associated with the different hydrolytic rates on YFV NS2B-NS3pro natural substrates.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
authors.forename: Marcia Y
authors.initials: MY
authors.lastname: Kondo
authors.forename: Lilian C G
authors.initials: LC
authors.lastname: Oliveira
authors.forename: Debora N
authors.initials: DN
authors.lastname: Okamoto
authors.forename: Marina R T
authors.initials: MR
authors.lastname: de Araujo
authors.forename: Claudia N
authors.initials: CN
authors.lastname: Duarte dos Santos
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
mesh_terms: D020816:Amino Acid Motifs; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010455:Peptides / Q000737:chemistry; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics; D015005:Yellow fever virus / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D010455:Peptides; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases