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Modeling of enzymatic reactions in vesicles: The case of α‐chymotrypsin
Author(s) -
Blocher Markus,
Walde Peter,
Dunn Irving J.
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19990105)62:1<36::aid-bit5>3.0.co;2-u
Subject(s) - vesicle , chemistry , aqueous solution , bilayer , substrate (aquarium) , chymotrypsin , popc , phospholipid , enzyme kinetics , hydrolysis , chromatography , lipid bilayer , aqueous two phase system , analytical chemistry (journal) , membrane , enzyme , organic chemistry , biochemistry , active site , trypsin , oceanography , geology
The kinetic behavior of the α‐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 α‐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 k cat and K S (respectively K M ) 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 × 10 −7 cm/s. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 36–43, 1999.