Ac-EVKKQR-pNA is a chromogenic dengue virus protease substrate derived from the NS2B/NS3 cleavage-site sequence. It contains the P6–P1 hexapeptide Ac-Glu-Val-Lys-Lys-Gln-Arg linked to p-nitroaniline for colorimetric detection of protease-mediated cleavage.
The substrate is particularly useful for studying how dengue NS2B-NS3 protease recognizes the unusual NS2B/NS3 junction compared with other viral polyprotein cleavage sites.
Product Information
| Property | Specification |
|---|---|
| Product Name | Ac-EVKKQR-pNA |
| Catalog No. | AS2525 |
| Sequence | Ac-Glu-Val-Lys-Lys-Gln-Arg-pNA |
| Sequence Shortening | Ac-EVKKQR-pNA |
| Peptide Length | 6 amino-acid residues |
| Molecular Formula | C41H58N14O12 |
| Molecular Weight | Approximately 949.09 Da |
| Reporter Group | p-Nitroaniline (pNA) |
| Research Enzyme | Dengue virus NS2B-NS3 protease |
| Cleavage-Site Context | NS2B/NS3 |
An Atypical Dengue Protease Cleavage Sequence
Many dengue NS2B-NS3 protease cleavage sites contain two basic residues immediately upstream of the scissile bond. The NS2B/NS3 junction is an important exception.
In Ac-EVKKQR-pNA, the P2 residue is Gln rather than Lys or Arg, while Arg occupies P1. This sequence feature makes the substrate useful for studying how the viral protease accommodates a cleavage site that differs from the more typical dibasic recognition pattern.
We think this distinction is important when comparing dengue protease substrates because peptides representing different viral junctions should not be assumed to have equivalent enzyme kinetics simply because they are cleaved by the same protease.
Published Enzyme Kinetics
In a recombinant dengue virus type 2 NS2B-NS3 protease study, Ac-EVKKQR-pNA showed a reported Km of approximately 1079 µM, kcat of approximately 0.077 s−1, and catalytic efficiency of approximately 72 M−1s−1 under the reported conditions.
Its Km was substantially higher than those reported for the 2A/2B and 3/4A pNA substrates in the same experiment, illustrating that natural cleavage-site origin does not guarantee identical affinity or turnover behavior in a short synthetic substrate format.
pNA-Based Detection
Cleavage releases p-nitroaniline, which can be followed by absorbance, commonly near 405 nm. This allows Ac-EVKKQR-pNA to be used in kinetic assays and inhibitor studies without a fluorescence-based detection system.
Researchers comparing substrate sequences should maintain consistent enzyme concentration, substrate range and reaction conditions because these variables can strongly influence apparent activity.
Experimental Planning
A published comparison of the P6–P1 pNA substrate with a longer NS2B/NS3 peptide extending through P4′ found that the longer construct was processed less efficiently under those conditions. This demonstrates that adding more residues around a natural cleavage site does not automatically improve a biochemical substrate.
We recommend selecting dengue protease substrates according to the experimental question: cleavage-site recognition, kinetic comparison, inhibitor screening or assay sensitivity.
Researchers developing alternative substrate sequences or reporter conjugates can use our Chemical Peptide Synthesis capabilities.
Frequently Asked Questions
What viral junction does Ac-EVKKQR-pNA represent?
The EVKKQR sequence corresponds to the P6–P1 region of the dengue NS2B/NS3 cleavage site.
Why is the P2 glutamine notable?
Many NS2B-NS3 protease cleavage sites contain a basic residue at P2. The NS2B/NS3 junction contains Gln at this position, giving it a distinct recognition sequence.
Can this substrate be used for inhibitor screening?
Yes. pNA release provides a direct protease-activity readout that can be incorporated into appropriately controlled inhibitor assays.