title: Product release is rate-limiting for catalytic processing by the Dengue virus protease.
abstract: Dengue Virus (DENV) is the most prevalent global arbovirus, yet despite an increasing burden to health care there are currently no therapeutics available to treat infection. A potential target for antiviral drugs is the two-component viral protease NS2B-NS3pro, which is essential for viral replication. Interactions between the two components have been investigated here by probing the effect on the rate of enzyme catalysis of key mutations in a mobile loop within NS2B that is located at the interface of the two components. Steady-state kinetic assays indicated that the mutations greatly affect catalytic turnover. However, single turnover and fluorescence experiments have revealed that the mutations predominantly affect product release rather than substrate binding. Fluorescence analysis also indicated that the addition of substrate triggers a near-irreversible change in the enzyme conformation that activates the catalytic centre. Based on this mechanistic insight, we propose that residues within the mobile loop of NS2B control product release and present a new target for design of potent Dengue NS2B-NS3 protease inhibitors.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: A E
authors.initials: AE
authors.lastname: Shannon
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: M M
authors.initials: MM
authors.lastname: Pedroso
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: K J
authors.initials: KJ
authors.lastname: Chappell
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: D
authors.initials: D
authors.lastname: Watterson
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Liebscher
authors.affiliation: ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: W M
authors.initials: WM
authors.lastname: Kok
authors.affiliation: ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: D P
authors.initials: DP
authors.lastname: Fairlie
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Schenk
authors.affiliation: ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: P R
authors.initials: PR
authors.lastname: Young
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003716:Dengue Virus / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000085242:Viral Proteases
chemical_list: D009842:Oligopeptides; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases