Modeling of enzymatic reactions in vesicles: the case of alpha-chymotrypsin.
The kinetic behavior of the alpha-chymotrypsin-catalyzed hydrolysis of the two p-nitroanilide substrates succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) and benzoyl-L-Tyr-p-nitroanilide (Bz-Tyr-pNA) was modeled and simulated for two different systems, namely for an aqueous solution and for a vesicle system, which was composed of phospholipid vesicles containing entrapped alpha-chymotrypsin. In the case of the vesicles, the substrate was added to the bulk, exovesicular aqueous phase. The experimentally determined time-dependence of product (p-nitroaniline) formation was modeled by considering the kinetic behavior of the enzyme and-in the case of vesicles-the substrate permeability across the bilayer membrane. In aqueous solution-without vesicles-the kinetic constants kcat and KS (respectively KM) were determined from fitting the model to experimental data of batch product concentration-time curves. The results were in good agreement with the corresponding values obtained from initial velocity measurements. For the vesicle system, using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), simulation showed that the substrate permeation across the bilayer was rate limiting. Using experimental data, we could obtain the substrate permeability coefficient for Bz-Tyr-pNA by parametric fitting as 2. 45 x 10(-7) cm/s.
Blocher
M
M
Institut für Polymere, ETH-Zentrum, Universitätstrasse 6, CH-8092 Zürich, Switzerland.
Walde
P
P
Dunn
I J
IJ
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D004800:Enzymes, Immobilized; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D008854:Microscopy, Electron; D008954:Models, Biological; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D008081:Liposomes; D002918:Chymotrypsin