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NLM AIDSLINE

Real-time measurements of dark substrate catalysis.




 

Protein Sci. 1999 Nov;8(11):2460-4. Unique Identifier : AIDSLINE

We have developed a novel procedure to monitor the real-time cleavage of natural unmodified peptides (dark substrates). In the competition-based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady-state enzyme kinetics for a two-substrate system, we were able to determine both Km and k(cat) values for cleavage of the dark substrate. The method was applied to HIV-1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.

JOURNAL ARTICLE Amino Acid Sequence Amino Acid Substitution Catalysis Enzymes/*METABOLISM HIV Protease/*CHEMISTRY/*METABOLISM Kinetics Models, Theoretical Mutagenesis, Site-Directed Oligopeptides/CHEMISTRY/METABOLISM Regression Analysis Substrate Specificity Support, U.S. Gov't, P.H.S.



 




Information in this article was accurate in March 30, 2000. The state of the art may have changed since the publication date. This material is designed to support, not replace, the relationship that exists between you and your doctor. Always discuss treatment options with a doctor who specializes in treating HIV.