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
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 mum). 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 mum for oleate and arachidonate, and 115-230 mum 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.
History
Publication title
The FEBS Journal
Volume
275
Issue
6
Pagination
1274-1282
ISSN
0945-5795
Department/School
Australian Maritime College
Publisher
Wiley-Blackwell Publishing Ltd.
Place of publication
United Kingdom
Rights statement
Copyright 2008 The Authors. Licensed under Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/
Repository Status
Open
Socio-economic Objectives
Expanding knowledge in the information and computing sciences