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-nitroanilid e (Bz-Tyr-pNA) was modeled and simulated for two different systems, na mely for an aqueous solution and for a vesicle system, which was compo sed of phospholipid vesicles containing entrapped ci-chymotrypsin, In the case of the vesicles, the substrate was added to the bulk, exovesi cular aqueous phase. The experimentally determined time-dependence of product (p-nitroaniline) formation was modeled by considering the kine tic behavior of the enzyme and-in the case of vesicles-the substrate p ermeability across the bilayer membrane. In aqueous solution-without v esicles-the kinetic constants k(cat) and K-S (respectively K-M) were d etermined from fitting the model to experimental data of batch product concentration-time curves. The results were in good agreement with th e corresponding values obtained from initial velocity measurements. Fo r the vesicle system, using the phospholipid 1-palmitoyl-2-oleoyl-sn-g lycero-3-phosphocholine (POPC), simulation showed that the substrate p ermeation across the bilayer was rate limiting. Using experimental dat a, we could obtain the substrate permeability coefficient for Bz-Tyr-p NA by parametric fitting as 2.45 x 10(-7) cm/s. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 36-43, 1999.