Suppressed catalytic efficiency of plasmin in the presence of long-chain fatty acids: Identification of kinetic parameters from continuous enzymatic assay with Monte Carlo simulation

Anna Tanka-Salamon, Kiril Tenekedjiev, Raymund Machovich, Krasimir Kolev

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Thrombi, which are dissolved primarily by plasmin (EC 3.4.21.7.), contain up to millimolar concentrations of fatty acids and these are known to affect the action of the protease. In the present study the modulation of plasmin activity was characterized quantitatively in a continuous amidolytic assay based on synthetic plasmin substrate (Spectrozyme-PL). A novel numerical procedure was applied for identification of kinetic parameters and their confidence intervals, with Monte Carlo simulation of the reaction progress curves, providing adequate grounds for discrimination of different models of the enzyme action. All three fatty acids caused a 10-20-fold increase in the Michaelis constant on Spectrozyme-PL (baseline value 5.9 μm). The catalytic constant decreased from 5.8·s-1 to 2.4-2.8·s-1 in the presence of arachidonate and oleate, but increased to 14.8·s-1 in the presence of stearate, implying enhancement of plasmin activity at saturating substrate concentrations. However, based on the ratio of the catalytic and Michaelis constants, all three fatty acids acted as inhibitors of plasmin with various degrees of potency, showing concentration dependence in the range of 10-65 μm for oleate and arachidonate, and 115-230 μm for stearate. The reported effects of the three fatty acids require the presence of kringle 5 in the structure of the protease; miniplasmin (des-kringle 1-4 plasmin) is as sensitive to fatty acids as plasmin, whereas the activity of microplasmin (des-kringle 1-5 plasmin) is not affected.

Original languageEnglish
Pages (from-to)1274-1282
Number of pages9
JournalFEBS Journal
Volume275
Issue number6
DOIs
Publication statusPublished - Mar 1 2008

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Keywords

  • Arachidonate
  • Monte Carlo simulation
  • Oleate
  • Progress curves
  • Stearate

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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