Product release is rate-limiting for catalytic processing by the Dengue virus protease

Area of Science:

• Virology
• Biochemistry
• Drug Discovery

Background:

• Dengue Virus (DENV) is a major global arbovirus with no available therapeutics.
• The DENV protease NS2B-NS3pro is essential for viral replication and a potential antiviral target.

Purpose of the Study:

• To investigate the role of interactions between the NS2B and NS3 components of the DENV protease.
• To understand how mutations in the NS2B mobile loop affect enzyme catalysis and identify new therapeutic targets.

Main Methods:

• Site-directed mutagenesis of the NS2B mobile loop.
• Steady-state and single-turnover kinetic assays.
• Fluorescence spectroscopy to analyze enzyme conformational changes and substrate binding.

Main Results:

• Mutations in the NS2B mobile loop significantly altered catalytic turnover rates.
• Experiments indicated that mutations predominantly affect product release, not substrate binding.
• Substrate addition induces a conformational change activating the catalytic center, influenced by the NS2B loop.

Conclusions:

• Residues in the NS2B mobile loop are critical for controlling product release in DENV protease activity.
• This mechanistic insight provides a novel target for designing potent Dengue NS2B-NS3 protease inhibitors.
• Targeting the product release mechanism could lead to effective antiviral strategies against Dengue virus.