Hydrolysis, phosphate, p-nitrophenyl, alkaline phosphatase, competitive inhibition
This study investigates the kinetics of p-nitrophenyl phosphate hydrolysis by mucosal alkaline phosphatase, emphasizing competitive inhibition. Using spectrophotometry at 405 nm, enzyme activity rates were determined across various substrate concentrations. The Lineweaver-Burk plot, transforming Michaelis-Menten data, was crucial for calculating Vmax, Km, and Ki. Results showed that sodium phosphate, a competitive inhibitor, altered Km but not Vmax, which was consistent with competitive inhibition characteristics. The inhibitor's potency was confirmed by a low Ki value. This research contributes to understanding enzyme behaviour in the presence of inhibitors, which is vital for biochemical studies and drug development.
[...] In the study of the hydrolysis of p-nitrophenyl phosphate (PNP) by mucosal alkaline phosphatase, spectrophotometry plays a crucial role. The enzyme alkaline phosphatase, which operates optimally at an alkaline pH, catalyzes the hydrolysis of various organic phosphates, including PNP. This reaction results in the release of p-nitrophenol, a bright yellow product. The reaction's progress can be monitored using a spectrophotometer set at 405 nm, the wavelength at which p-nitrophenol has its maximum absorbance. By constructing a plot of absorbance at 405 nm (A405) against time, the rate of the reaction can be quantified. [...]
[...] Since this is not always the case, the actual rate of reaction is determined by the initial ratio of enzyme to substrate. In a reaction wherein the substrate concentration is more than the enzyme concentration, the rate of reaction is lowered due to the restraint posed by the lower enzyme concentration. The maximum reaction rate is achieved when the enzyme and substrate start at an equal concentration (Kargi, 2009). The Lineweaver-Burk plot is a graphical representation used to analyze the kinetics of enzyme-catalysed reactions, particularly in situations where the Michaelis-Menten equation is applied. [...]
[...] The value of is 1mM, since that is the concentration of the inhibitor. Results: Figure 7 Time Courses for Sodium Phosphate. Plotted Absorbance vs Time (Seconds) Tube Numbers Time (seconds) 30 60 90 120 150 180 T7 0.208 0.425 0.621 0.823 1.014 1.226 T6 0.170 0.360 0.534 0.703 0.886 1.070 T5 0.149 0.281 0.422 0.572 0.712 0.845 T4 0.122 0.236 0.359 0.479 0.594 0.708 T3 0.088 0.173 0.256 0.351 0.449 0.543 T2 0.062 0.125 0.178 0.250 0.337 0.419 T1 0.034 0.073 0.110 0.150 0.185 0.223 Table Absorption Values for Sodium Phosphate at 405nm for all 7 Tubes. [...]
[...] It is defined as the concentration of an inhibitor required to reduce the enzyme activity to half its standard rate in the absence of the inhibitor. A lower Ki value represents a potent inhibitor, which in this experiment holds true; with a value of 0.54, we can state that the inhibitor used was potent. References: Kakkar, T., Boxenbaum, H. and Mayersohn, M. (1999) Estimation of in a competitive enzyme-inhibition model: Comparisons among three methods of data analysis, Drug Metabolism and Disposition, 27(6). [...]
[...] Figure 1 represents the plotting of time courses for sodium phosphate, and Figure 2 represents the plotting of time courses for distilled Water. The gradient/slope for each line of best fit drawn on both graphs (figure 1 and was calculated and tabulated in Tables 2 and 4. The gradient values represent the enzyme's initial velocity, plotted in Figure 3 on the y-axis against the substrate concentrations on the x-axis. Since the initial velocities were very small, they were multiplied by 1000 to make plotting more convenient. [...]
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