Enzyme Activity Lecture. Every reaction has energy requirement. The minimum amount of energy required is termed activation energy.

Transcription

1 Enzyme Activity Lecture Every reaction has energy requirement. The minimum amount of energy required is termed activation energy. Living organisms have optimum temperature requirement so elevating the temperature to increase incidence of molecular collision will harm the biological structure. Increasing the concentration of the reactants also increases the likelihood of collisions, however concentrations are normally low. Enzymes are used by biological systems to lower the activation energy. The enzymes provide another pathway that requires less energy and increases the rate of reaction. Enzyme Properties: 1. Show remarkable specificity for their reactants, called substrates. 2. Nearly all enzymes are protein. 3. Each enzyme has a unique, intricately shaped binding surface called the active site. 4. Most enzymes often contain non-protein components such as metal ions called cofactor. Co-enzymes are organic nom-protein component (example: vitamins) 5. The protein component of an enzyme is called the apoenzyme and an enzyme that has the cofactor intact is called the haloenzyme. 6. The non-covalent forces that bind substrate to the active site are the same forces that account for the conformations of protein themselves: van der Waals forces, electrostatic forces, hydrogen bonding and hydrophobic interactions.

2 7. How do enzymes work? The Induced Fit Model. The enzyme and the substrate combine to form an enzyme-substrate complex. The formation of the complex often induces a conformational change in that allows it to bind to the substrate. Binding the substrate also often causes certain of its bond to become strained, and therefore is easily broken. The product of the reaction usually has a different shape from the substrate, and the altered shape or the intervention of another molecule causes the complex to dissociate. The enzyme can then accept another molecule of substrate, repeating the whole process. 1. How is enzyme activity regulated? The reaction is stopped by enzyme inhibition: competitive, non-competitive and feedback inhibition. In competitive inhibition, another substance other than the substrate competes for the active site of the enzyme. This type of inhibition slows down the reaction and is reversible.

3 In non-competitive inhibition, the inhibitors with portions of the active site which results in the changing of its shape. Once the active site's shape is changed, it can no longer attach to the substrate In allosteric inhibition another substance bind to the enzyme in a spot other than the active site, altering the conformation of the enzyme. The enzyme is left non-functional.

4 In feedback inhibition, the amount of product present or absent determines enzyme activity. 2. Factors that affect the enzyme activity. a. Substrate Concentration Rate of reaction initially increases as substrate concentration is increased. At a certain concentration the substrate, the rate of reaction reaches a maximum. b. Enzyme Concentration Rate of reaction increases with increased enzyme concentration. There will be a leveling off as the substrate is used up. 3. Effect of Temperature and ph Enzymes function most effectively at neutral ph. The rate of reaction falls rapidly as the solution is made acidic or alkaline. At extreme ph the enzyme actually looses it biological active conformation and is denatured. Enzymes are rapidly destroyed if the temperature rise above 37 o C but remain stable at much lower temperatures.

5 Experiment: Part A Extraction of polypheloxidase Extract polyphenoloxidase from potato. Polyphenoloxidase catalyses oxidation of dihydroxyphenols to quinones. Colorless Reddish Brown Part B Enzyme specificity Will compounds that have similar structure as cathechol also be catalyzed by polyphenoxidase? Phenol 1,4 cyclohexanediol Cathecol

6 For the remainder of the experiment: Keep test tube A from Part B as your negative control. Keep test tube B from Part B as your positive control Part C Substrate Concentration Because the enzyme extracted for the potato is not quantified, a qualitative measure is done. You must be able to differentiate the color among the different enzyme concentrations. If no difference in intensity is observed in Part C, the enzyme might be too concentrated. Dilute enzyme stock solution 2:1 (8 ml enzyme + 4 ml 2% NaF) and repeat the Part C with the diluted enzyme. If this is still too concentrated, dilute the enzyme 1:1 with the NaF solution. Once the optimum concentration is achieved, use the same enzyme concentration for the remaining parts of the experiment. Part D Enzyme Concentration Part E Effect of ph Part F Effect of Temperature Part G Inhibitors When a ppt is observed, the protein has denatured. Phenylthiourea combines with the copper cofactor. Is it a competitive or noncompetitive inhibitor. Data Analysis Recommendation: Graph with Excel. Determine the relative intensity among the samples for each part of the experiment only. What do Parts B and G determine. Plot graph for Part C and D Part C plot Intensity VS Relative Substrate Concentration Part D plot Intensity VS Relative Enzyme Concentration Connect the points in the graph. Do not do a best fit. For Part E and F A bar graph would represent the data best.

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