6.3 Results and Discussion
6.3.1 The influence of high pressure on the kinetics of α-chymotrypsin
The choice was determined by the fact that the catalytic mechanism as well as pressure stability of this enzyme is well established. α-chymotrypsin-catalyzed hydrolysis proceed according to the Michaelis–Menten mechanism. The initial process involves creation of a stable enzyme substrate complex ES, which is related to KM, afterwards activation of the ES^≠ and subsequent reaction steps occur, which are governed by the catalytic rate constant kcat. As a model peptide anilide substrate for hydrolysis SAAPPpNA (N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanine-p-nitroanilide) was selected. Reaction was observed as a function of time by monitoring increment of absorbance data at 410nm as a result of the p-nitroaniline product formation. Initial reaction rate vo was determined by converting raw absorption data units to moles per second using molar extinction coefficient of the substrate. Reaction rates were plotted as a function of substrate concentration to obtain Michaelis-Menten plot. Lineweaver - Burk plot was built to obtain kinetic parameters KM and kcat. The activation volume 〖ΔV〗^≠was derived from the slope of logarithmic Vmax dependence on pressure p. The raw data, Michaelis-Menten plot, The double reciprocal plot, i.e, Lineweaver - Burk plot and logarithmic plot of vmax (in relation to the rate at ambient pressure) as a function of pressure for the hydrolysis of the substrate by α-chymotrypsin are …show more content…
With increasing pressure KM is nearly unchanged, indicating the binding volume, ΔVES≈0, in buffer solution, while kcat increases linearly, resulting a negative value of activation volume of reaction. The activation volume calculated from the slope of the curve in Figure 2d using equation 4.9 yields to -11±1 mL/mol that corresponds to a 2-fold reaction rate increase at a pressure
6.3.1 The influence of high pressure on the kinetics of α-chymotrypsin
The choice was determined by the fact that the catalytic mechanism as well as pressure stability of this enzyme is well established. α-chymotrypsin-catalyzed hydrolysis proceed according to the Michaelis–Menten mechanism. The initial process involves creation of a stable enzyme substrate complex ES, which is related to KM, afterwards activation of the ES^≠ and subsequent reaction steps occur, which are governed by the catalytic rate constant kcat. As a model peptide anilide substrate for hydrolysis SAAPPpNA (N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanine-p-nitroanilide) was selected. Reaction was observed as a function of time by monitoring increment of absorbance data at 410nm as a result of the p-nitroaniline product formation. Initial reaction rate vo was determined by converting raw absorption data units to moles per second using molar extinction coefficient of the substrate. Reaction rates were plotted as a function of substrate concentration to obtain Michaelis-Menten plot. Lineweaver - Burk plot was built to obtain kinetic parameters KM and kcat. The activation volume 〖ΔV〗^≠was derived from the slope of logarithmic Vmax dependence on pressure p. The raw data, Michaelis-Menten plot, The double reciprocal plot, i.e, Lineweaver - Burk plot and logarithmic plot of vmax (in relation to the rate at ambient pressure) as a function of pressure for the hydrolysis of the substrate by α-chymotrypsin are …show more content…
With increasing pressure KM is nearly unchanged, indicating the binding volume, ΔVES≈0, in buffer solution, while kcat increases linearly, resulting a negative value of activation volume of reaction. The activation volume calculated from the slope of the curve in Figure 2d using equation 4.9 yields to -11±1 mL/mol that corresponds to a 2-fold reaction rate increase at a pressure