Thermal and pH effects on stromelysin kinetics

C. M. Colvis, Ted Acott

Research output: Contribution to journalArticle

Abstract

Purpose. We hypothesize that matrix metalloproteinase (MMP) activity, particularly that of stromelysin, is responsible for maintaining aqueous humor outflow facility by initiating extracellular matrix turnover directly and by activating other members of the MMP family. The kinetic mechanism of stromelysin is only partially understood. Methods. Purified stromelysin was subjected to kinetic analysis using fluorescein isothiocyanate labeled protein substrate and a quench-release fluorescent peptide substrate (NFF3). Substrate concentration, temperature and pH were varied and linear and non-linear curve fitting were used to analyze the data. Results. At neutral pH, non-Michaelis/Menton kinetic and non-linear thermodynamic curves were obtained with both protein and peptide substrates, suggesting a multistep kinetic mechanism. To differentiate between a sequential and one type of parallel multistep mechanism, thermal and substrate dependencies were evaluated at higher and lower pH values. This simplified the kinetics curves, allowing us to evaluate apparent single-step mechanisms. Conclusion. A parallel multistep mechanism had been proposed for stromelysin, suggesting two functional forms of the enzyme. Our data supports such a parallel multistep model with the two functional forms differing in their protonation state. By using two types of substrates, we eliminate substrate as a source of the non-linear behavior. Thus, modifying reaction pH allows us to evaluate the two enzyme forms independently.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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Matrix Metalloproteinase 3
Hot Temperature
Matrix Metalloproteinases
Peptides
Aqueous Humor
Enzymes
Fluorescein
Thermodynamics
Extracellular Matrix
Proteins
Temperature

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Thermal and pH effects on stromelysin kinetics. / Colvis, C. M.; Acott, Ted.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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