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.
Tanka-Salamon
Anna
A
Department of Medical Biochemistry, Semmelweis University, Puskin u.9, Budapest, Hungary.
Tenekedjiev
Kiril
K
Machovich
Raymund
R
Kolev
Krasimir
K
D016718:Arachidonic Acid / Q000737:chemistry*; D002384:Catalysis; D003198:Computer Simulation; D005341:Fibrinolysin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009010:Monte Carlo Method; D019301:Oleic Acid / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry; D013228:Stearates / Q000737:chemistry*; D013329:Structure-Activity Relationship
D009842:Oligopeptides; D013228:Stearates; D016718:Arachidonic Acid; D019301:Oleic Acid; C060237:H-norleucyl-hexahydrotyrosyl-lysine-4-nitroanilide; D005341:Fibrinolysin