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 (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 | 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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Hepacivirus C | - |
- |
- |
Hepacivirus C | Q9WPH5 | subtype 1b | - |
Purification (Comment) | Organism |
---|---|
gel filtration | Hepacivirus C |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
helicase assay at | Hepacivirus C |
37 | - |
ATPase and RNA binding assay at | Hepacivirus C |
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 |