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Literature summary for 3.6.4.13 extracted from

  • Beran, R.K.; Lindenbach, B.D.; Pyle, A.M.
    The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase (2009), J. Virol., 83, 3268-3275.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
nonstructural protein 4A i.e. NS4A, enhances the coupling between RNA binding and ATPase activity of nonstructural protein 3 (NS3), does not influence the kinetic parameters for RNA unwinding by NS3 Hepacivirus C
nonstructural protein 4A NS4A binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity, thus NS4A enhances the ability of the C-terminal helicase to bind RNA in presence of ATP acting as a cofactor for helicase activity, 100fold lower Km of NS3 with RNA in presence of NS4A. NS4A mutants that are defective in ATP-coupled RNA binding sre lethal in vivo Hepacivirus C

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant NS3, cloning of a His6-tag to the N-terminus of NS3 greatly increases its affinity for RNA Hepacivirus C
NS3-plus and NS3/4a-plus genes expressed in Escherichia coli, composition of NS3-4A expression product using the pet-SUMO vector Hepacivirus C

Protein Variants

Protein Variants Comment Organism
M1708A NS3-4A construct, ability to bind and unwind RNA in vitro, mutation reduces functional NS3-4A binding affinity for RNA by 500-fold relative to the wild-type Hepacivirus C
M1708A site-directed mutagenesis, the NS3-4A mutant shows decreased ATPase activity and reduced RNA stimulation activity compared to wild-type NS3 Hepacivirus C
additional information construction of the NS3-4A mutant affected in its acidic domain, the mutant shows altered RNA binding and increased ATPase activity, kinetics, overview Hepacivirus C
S1369R NS3-4A construct, suppressor mutant, ATP-coupled RNA affinity identical to that of wild-type NS3-4A Hepacivirus C
S1369R site-directed mutagenesis, the NS3-4A mutant shows increased ATPase activity and RNA stimulation activity compared to wild-type NS3 Hepacivirus C
S1369R/M1708A NS3-4A construct, reduced ATP-coupled RNA affinity of the single mutant suppressed by the addition of the S1369R mutation Hepacivirus C
S1369R/M1708A site-directed mutagenesis, the NS3-4A mutant shows increased ATPase activity and reduced RNA stimulation activity compared to wild-type NS3 Hepacivirus C
S1369R/Y1702A NS3-4A construct, reduced ATP-coupled RNA affinity of the single mutant suppressed by the addition of the S1369R mutation Hepacivirus C
S1369R/Y1702A site-directed mutagenesis, the NS3-4A mutant shows decreased ATPase activity and reduced RNA stimulation activity compared to wild-type NS3 Hepacivirus C
Y1702A NS3-4A construct, ability to bind and unwind RNA in vitro, mutation reduces functional NS3-4A binding affinity for RNA by 500-fold relative to the wild-type Hepacivirus C
Y1702A site-directed mutagenesis, the NS3-4A mutant shows decreased ATPase activity and reduced RNA stimulation activity compared to wild-type NS3 Hepacivirus C

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics of wild-type and mutant enzymes, overview Hepacivirus C
additional information
-
additional information RNA-stimulated ATPase activities of NS3-4A variants analyzed, functionally important ATP-bound state of NS3 binds RNA much more tightly in the presence of NS4A, effectively coupling RNA binding to ATPase activity Hepacivirus C
0.000001
-
ATP pH 6.5, 37°C, RNA-stimulated ATPase activity of mutant NS3-4A Hepacivirus C
0.000002
-
ATP pH 6.5, 37°C, RNA-stimulated ATPase activity of mutant S1369R/M1708A Hepacivirus C
0.0001
-
ATP pH 6.5, 37°C, RNA-stimulated ATPase activity of wild-type NS3 Hepacivirus C
0.0005
-
ATP pH 6.5, 37°C, RNA-stimulated ATPase activity of mutant M1708A Hepacivirus C
0.0005
-
ATP pH 6.5, 37°C, RNA-stimulated ATPase activity of mutant Y1702A Hepacivirus C
0.001
-
ATP RNA-stimulated ATPase activity, recombinant protein, NS3-4A construct Hepacivirus C
0.001
-
ATP RNA-stimulated ATPase activity, recombinant protein, NS3-4A S1369R mutant Hepacivirus C
0.002
-
ATP RNA-stimulated ATPase activity, recombinant protein, NS3-4A S1369R/M1708A mutant Hepacivirus C
0.03
-
ATP RNA-stimulated ATPase activity, recombinant protein, NS3-4A S1369R/Y1702A mutant Hepacivirus C
0.05
-
ATP RNA-stimulated ATPase activity, recombinant protein, NS3-4A M1708A mutant Hepacivirus C
0.05
-
ATP RNA-stimulated ATPase activity, recombinant protein, NS3-4A Y1702A mutant Hepacivirus C
0.1
-
ATP RNA-stimulated ATPase activity, NS3, recombinant protein Hepacivirus C

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + H2O Hepacivirus C RNA-stimulated ATPase activities determined, interaction between the replicative component nonstructural protein 3 (NS3) with the nonstructural protein 4A (NS4A) ADP + phosphate
-
?
additional information Hepacivirus C the C-terminal region of NS3 exhibits RNA-stimulated NTPase, e.g. ATPase, and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by the C-terminal region, NS4A mutants that are defective in ATP-coupled RNA binding are lethal in vivo ?
-
?

Organism

Organism UniProt Comment Textmining
Hepacivirus C
-
-
-
Hepacivirus C Q9WPH5 subtype 1b
-

Purification (Commentary)

Purification (Comment) Organism
gel filtration Hepacivirus C

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
the nonstructural protein 4A (NS4A) enhances the ability of the N-terminal domain of NS3 protein to bind RNA in the presence of ATP, stimulates helicase activity, interaction between nonstructural protein 3 (NS3) and nonstructural protein 4A (NS4) mediated by amino acids of the C-terminus of NS4, mutation of the C-terminus of NS4 reduces ATP-coupled RNA binding, RNA binding studies, RNA-stimulated ATPase activity of N3-4a variants Hepacivirus C

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + H2O
-
Hepacivirus C ADP + phosphate
-
?
ATP + H2O RNA-stimulated ATPase activities determined, interaction between the replicative component nonstructural protein 3 (NS3) with the nonstructural protein 4A (NS4A) Hepacivirus C ADP + phosphate
-
?
additional information the C-terminal region of NS3 exhibits RNA-stimulated NTPase, e.g. ATPase, and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by the C-terminal region, NS4A mutants that are defective in ATP-coupled RNA binding are lethal in vivo Hepacivirus C ?
-
?
RNA + H2O unwinding helicase activity, NS3 is ahighly basic protein with multiple RNA binding sites Hepacivirus C ?
-
?

Synonyms

Synonyms Comment Organism
nonstructural protein 3
-
Hepacivirus C
nonstructural protein 3 ambiguous Hepacivirus C
NS3 ambiguous Hepacivirus C
NS3 helicase
-
Hepacivirus C

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
helicase assay at Hepacivirus C
37
-
ATPase and RNA binding assay at Hepacivirus C

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
helicase assay at Hepacivirus C
6.5
-
ATPase and RNA binding assay at Hepacivirus C