Related Experiment Videos
Stabilization of HIV proteinase dimer by bound substrate
P Kuzmic1, C García-Echeverría, D H Rich
1School of Pharmacy, University of Wisconsin, Madison 53706.
Biochemical and Biophysical Research Communications
|July 15, 1993
Summary
The binding of substrate stabilizes the active dimeric form of HIV proteinase, preventing dissociation into inactive monomers. This substrate-induced stabilization impacts HIV proteinase activity measurements in kinetic studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- Human Immunodeficiency Virus (HIV) proteinase is a crucial enzyme for viral replication, existing as an active dimer.
- Understanding the stability and dissociation of HIV proteinase is essential for developing effective antiviral therapies.
Purpose of the Study:
- To investigate the effect of synthetic nonapeptide substrate binding on the stability of the catalytically active dimeric form of HIV proteinase.
- To quantify the dissociation constants of the enzyme-substrate complex and the enzyme dimer under specific conditions.
Main Methods:
- Enzyme kinetics assays were performed to measure the dissociation constants.
- Studies involved varying the order of addition of enzyme and substrate to assess activity dependence.
- Equilibrium dissociation constants were determined at pH 4.7 and ionic strength 1.0 M.
Main Results:
- Binding of a synthetic nonapeptide substrate strongly stabilizes the dimeric HIV proteinase against dissociation.
- The dissociation of the ternary Michaelis complex into monomers is immeasurably low (picomolar range).
- The dimer-to-monomer equilibrium dissociation constant was determined to be 30.4 +/- 1.6 nM at pH 4.7 and 1.0 M ionic strength.
Conclusions:
- Substrate binding significantly enhances the stability of the active HIV proteinase dimer.
- The apparent activity of HIV proteinase is influenced by the pre-incubation order of enzyme and substrate.
- Substrate-induced stabilization must be considered in kinetic studies of dissociative retroviral enzymes like proteinase, integrase, and reverse transcriptase.