Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 3.4.21.91 extracted from

  • Hill, M.E.; Yildiz, M.; Hardy, J.A.
    Cysteine disulfide traps reveal distinct conformational ensembles in dengue virus NS2B-NS3 protease (2019), Biochemistry, 58, 776-787 .
    View publication on PubMed
Search for more literature for 3.4.21.91

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of codon-optimized N-terminally His6-tagged dengue virus serotype 2 protease (DENV2 NS2B-NS3pro)from linked and unlinked constructs in Escherichia coli strain BL21(DE3) Dengue virus type 2

Protein Variants

Protein Variants Comment Organism
A125C/V162C site-directed mutagenesis, the mutant shows 32% reduction in activity due to the cysteine substitution. Linked A125C/V162C, in which both mutations are within the protease domain, does not migrate differently without DTT. The unlinked either trap does not form intermolecular disulfide bonds Dengue virus type 2
I73C/P106C site-directed mutagenesis, the linked open trap mutant does not form intermolecular disulfide bonds appreciably, as evidenced by the lack of higher-molecular weight bands. Unlinked I73C/P106C forms a small amount of NS2BS-SNS3pro, which shows retarded migration as NS2B and NS3pro electrophorese as a single disulfide-bonded complex Dengue virus type 2
K117A site-directed mutagenesis Dengue virus type 2
K117C site-directed mutagenesis Dengue virus type 2
K117R site-directed mutagenesis Dengue virus type 2
additional information multiple types of protein constructs have been used for NS2B-NS3pro expression. In one type, a Gly4SerGly4 linker joins NS2B to NS3pro, reported to improve expression and purification, while another type requires co-expression of the unlinked NS2B and NS3 regions. The catalytic activity is slightly different between the linked and unlinked protein constructs, with the unlinked form showing activity approximately 0-5fold greater than that of the linked form, likely due to constraints introduced by the synthetic linker Dengue virus type 2
S75C site-directed mutagenesis Dengue virus type 2
S75C/K117C site-directed mutagenesis, linked S75C/K117C is induced to form the NS2BS-SNS3pro closed conformation in the presence of active site inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that the mutation forms a disulfide bond when the enzyme is resting in the active conformation. The S75C/K117C pair is close together in the closed conformation but far apart in the open conformation and should therefore trap this variant in the closed conformation upon intramolecular disulfide bond formation Dengue virus type 2

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics of cysteine-substituted mutant variants Dengue virus type 2
0.03
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant A125C/V162C Dengue virus type 2
0.03
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant S75C/K117C Dengue virus type 2
0.04
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant I73C/P106C Dengue virus type 2
0.04
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117A Dengue virus type 2
0.04
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant S75C Dengue virus type 2
0.04
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant S75C/K117C Dengue virus type 2
0.04
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form wild-type enzyme Dengue virus type 2
0.05
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117C Dengue virus type 2
0.05
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117R Dengue virus type 2
0.05
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant A125C/V162C Dengue virus type 2
0.05
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant I73C/P106C Dengue virus type 2
0.07
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form wild-type enzyme Dengue virus type 2
0.08
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant K117A Dengue virus type 2

Organism

Organism UniProt Comment Textmining
Dengue virus type 2 P29990 DENV-2
-
Dengue virus type 2 Thailand/16681/1984 P29990 DENV-2
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged protease DENV2 NS2B-NS3pro linked and form from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, recombinant unlinked enzyme is purified by anion exchange chromatography Dengue virus type 2

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin + H2O
-
 Dengue virus type 2 N-acetyl-Gly-Arg-Arg + 7-amino-4-methylcoumarin
-
 ?
N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin + H2O
-
 Dengue virus type 2 Thailand/16681/1984 N-acetyl-Gly-Arg-Arg + 7-amino-4-methylcoumarin
-
 ?

Synonyms

Synonyms Comment Organism
dengue virus protease
-
 Dengue virus type 2
dengue virus serotype 2 protease
-
 Dengue virus type 2
DENV2 NS2BNS3pro
-
 Dengue virus type 2
NS2B-NS3pro
-
 Dengue virus type 2

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Dengue virus type 2

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.006
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117A Dengue virus type 2
0.01
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant A125C/V162C Dengue virus type 2
0.02
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant A125C/V162C Dengue virus type 2
0.02
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117C Dengue virus type 2
0.02
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant S75C/K117C Dengue virus type 2
0.03
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant I73C/P106C Dengue virus type 2
0.04
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117R Dengue virus type 2
0.05
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant S75C Dengue virus type 2
0.05
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form wild-type enzyme Dengue virus type 2
0.05
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant K117A Dengue virus type 2
0.07
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant S75C/K117C Dengue virus type 2
0.08
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant I73C/P106C Dengue virus type 2
0.09
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form wild-type enzyme Dengue virus type 2

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
 assay at Dengue virus type 2

Cofactor

Cofactor Comment Organism Structure
NS2B cofactor protein required for proteolytic activity Dengue virus type 2

General Information

General Information Comment Organism
additional information the protease is a two-component system such that a portion of NS2B in addition to the protease portion of NS3 is also required for proteolytic activity. The enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the closed conformation dramatically increases the activity, strongly suggesting that the closed conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an unlinked construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a closed, active conformation. In contrast, in a linked construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the closed and other alternative conformations. 4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF) induction of the closed conformation. Structure-function analysis, overview Dengue virus type 2
physiological function enzyme NS2B-NS3pro has a critical role in polyprotein maturation and viral infectivity Dengue virus type 2

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.44
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form wild-type enzyme Dengue virus type 2
0.57
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant S75C/K117C Dengue virus type 2
0.625
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant I73C/P106C Dengue virus type 2
0.71
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form wild-type enzyme Dengue virus type 2
0.8
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant S75C Dengue virus type 2
1.25
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117R Dengue virus type 2
1.33
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant I73C/P106C Dengue virus type 2
1.5
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant A125C/V162C Dengue virus type 2
1.5
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant S75C/K117C Dengue virus type 2
1.6
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant K117A Dengue virus type 2
2.5
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117C Dengue virus type 2
5
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant unlinked form mutant A125C/V162C Dengue virus type 2
6.67
-
 N-acetyl-Gly-Arg-Arg-7-amido-4-methylcoumarin pH 8.5, 37°C, recombinant linked form mutant K117A Dengue virus type 2