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Lab Activity4

This lab activity explores enzyme kinetics, focusing on factors that influence enzymatic reactions such as substrate concentration, temperature, inhibitors, activators, and pH. The experiment specifically studies the enzyme alkaline phosphatase (ALP) using a spectrophotometer assay.

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Lab Activity4

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  1. Lab Activity4 IUG, 2012 Dr. Tarek M. Zaida

  2. Enzyme Kinetics

  3. Enzymes • Enzyme reaction: E + S ↔ ES → E + P Whereas: E: Enzyme, S: Substrate, ES: Enzyme-Substrate complex, P: Product

  4. Enzymes decrease activation energy • A chemical reaction goes through a transition state with a higher G than either S of P • Enzymes facilitate the formation of the transition state by decreasing G‡

  5. Enzyme Kinetics • Factors influencing an enzymatic reaction • Substrate concentration • Temperature • Inhibitors • Activators • pH

  6. 1. Substrate Concentrate • At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S]. • But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola). • The asymptote represents the maximum velocity of the reaction, designated Vmax

  7. The substrate concentration that produces a Vi that is one-half of Vmaxis designated the Michaelis-Menten constant,Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).

  8. Allosteric effectors • Noncovalently bind and regulate the enzyme. • The effector may be stimulatory or inhibitory. • The substrate and effector usually occupy different specific binding sites.

  9. Enzyme Kinetics of Alkaline Phosphatase (ALP)

  10. Background • Function • R-PO4 + H2O → R-OH + H3PO4 • substrate product product The reaction catalyzed by alkaline Phosphatase

  11. Experiment • Facts.. • An assay is necessary to study an enzyme • The assay is a measurement of a chemical reaction, which might involve measuring the formation of the product (or otherwise the decrease in substrate conc.)

  12. Reagents and instruments • 18 labeled plastic tubes • Micropipette • Spectrophotometer • ALP enzyme kit (ready to use) • Blood serum • 5 N NaOH solution

  13. Procedure • Take 18 clean plastic tubes and label them from 1 to 18. Another tube will be used as a blank. This will contain all the reagents except of the enzyme. • Make the substrate and buffer concentrations as described in the given table (make sure to keep the total volume of all tubes stable at 1.9 ml). • Transfer 100 µl of serum to each tube. • Mix substrate and serum solutions and incubate at 37c for 50 seconds. • Add 0.5 ml of 5 N NaOH in each tube to stop the reaction. • Read the absorbance at 400 nm. • Plot reaction rate (Vi) on Y axis against substrate concentration [S] on X axis.

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