Chymotrypsin kinetic analysis

Detailed kinetic measurements and kinetic analysis (by dynamic modeling) of chymotrypsin-containing POPC vesicles. 

Kinetic analysis has even been attempted for the degradation of peptide and protein pharmaceuticals for which the mechanism and pathways are unknown. Apparent inactivation of a-chymotrypsin and bromelain in aqueous solutions was described by monoexponential... 

Figure 2 Time-resolved ESI-TOF-MS. (a) Diagram with the experimental design, (b) Chymotrypsin reaction with p-nitrophenyl acetate under single-turnover conditions monitored by time-resolved ESI-TOF-MS. The decay of chymotrypsin and formation of the acetyl-chymotrypsin intermediate is observed over a time course of 45 s. (c) Kinetic analysis of chymotrypsin decay and acetyl-chymotrypsin formation. For both traces the rate constant was 0.1 s . ...

If the a-chymotrypsin-catalysed hydrolysis of 4-nitrophenyl acetate [10] is monitored at 400 nm (to detect 4-nitrophenolate ion product) using relatively high concentrations of enzyme, the absorbance time trace is characterised by an initial burst (Fig. 5a). Obviously the initial burst cannot be instantaneous and if one uses a rapid-mixing stopped-flow spectrophotometer to study this reaction, the absorbance time trace appears as in Fig. 5b. Such observations have been reported for a number of enzymes (e.g. a-chymotrypsin [11], elastase [12], carboxypeptidase Y [13]) and interpreted in terms of an acyl-enzyme mechanism (Eqn. 7) in which the physical Michaelis complex, ES, reacts to give a covalent complex, ES (the acyl-enzyme) and one of the products (monitored here at 400 nm). This acyl-enzyme then breaks down to regenerate free enzyme and produce the other products. The dissociation constant of ES is k2 is the rate coefficient of acylation of the enzyme and A 3 is the deacylation rate coefficient. Detailed kinetic analysis of this system [11] has shown... 

This equation is fundamental to all aspects of the kinetics of enzyme action. The Michaelis-Menten constant, KM, is defined as the concentration of the substrate at which a given enzyme yields one-half of its maximum velocity. is the maximum velocity, which is the rate approached at infinitely high substrate concentration. The Michaelis-Menten equation is the rate equation for a one-substrate enzyme-catalyzed reaction. It provides the quantitative calculation of enzyme characteristics and the analysis for a specific substrate under defined conditions of pH and temperature. KM is a direct measure of the strength of the binding between the enzyme and the substrate. For example, chymotrypsin has a Ku value of 108 mM when glycyltyrosinylglycine is used as its substrate, while the Km value is 2.5 mM when N-20 benzoyltyrosineamide is used as a substrate... 

Banerjee, S. K. (1979). Humin fraction of soil humus. J. Indian Chem. Soc. 56,1094-1097. Baron, M. H., Revault, M., Servagent-Noinville, S., Abadie, J., and Quiquampoix, H. (1999). Chymotrypsin adsorption on montmorillonite Enzymatic activity and kinetic FTIR structural analysis. I. Coll. Interface Sci. 214, 319-332. 

Farber, G.K. (1999) Crystallographic analysis of solvent-trapped intermediates of chymotrypsin, in Schramm, V. L. and Punch, D. L. (eds.), Methods in Enzymology 308, Enzyme kinetics and Mechanism, Part E, Academic Press, San Diego, pp. 201-218. 

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