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Lab #5 info - Laboratory V Topic Enzyme Kinetics Principle...

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Laboratory V Topic : Enzyme Kinetics Principle : All enzymes show distinct kinetic features. During the initial period of an enzyme reaction, there is an excess of enzyme as compared to the substrate. Hence, the reaction velocity increases dependent on the substrate concentration. This is described as the initial velocity . As the reaction proceeds, all the enzyme sites get occupied until the velocity reaches a maximum velocity ( Vmax ). The reaction then continues at a steady state at the maximum velocity. If we plot a graph of the velocity of the enzyme reaction (V 0 ) vs the enzyme substrate [S] using a constant enzyme concentration , it appears as a hyperbola. This plot is mathematically described by Michaelis Menten equation and useful to compare the activity of enzymes. The Km is determined by drawing a Michaelis Menten graph or using the Michaelis Menten equation. The concentration of the substrate that will produce half the Vmax is known as Km . An enzyme with smaller Kms is more active than enzymes with higher Km values. As the substrate is used up, its concentration decreases, and the velocity of the reaction begins to slow down. In today’s exercise, we will look at the kinetics of Acid phosphatase enzyme. Enzyme acid phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate disodium (p-NPPO4 Na 2 ). The enzymatic reaction produces p-nitrophenol (p-NP) and phosphate. The enzyme works optimally in an acidic pH (pH 4.8) and at 37 ° C. Activity 1: Hydrolysis of p-NPP using acid Phosphatase enzyme .You will use a fixed concentration of the enzyme and vary the concentrations of the substrate p-nitrophenyl phosphate. The reaction will be stopped in 30 mins using NaOH. We measure the absorbance of p-nitrophenol formed at 400 nm.
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