Factors Affecting Enzymatic Activity PDF

Factors affecting enzymatic activity Yasmine M. Nageeb Ass. Lecturer of medical biochemistry Students’ learning outcomes By the end of this lecture, the students should be able to: 1. Define the term 'enzyme’ 2. Name and describe different factors that control enzyme...

Factors affecting enzymatic activity Yasmine M. Nageeb Ass. Lecturer of medical biochemistry Students’ learning outcomes By the end of this lecture, the students should be able to: 1. Define the term 'enzyme’ 2. Name and describe different factors that control enzyme activity. 3. Describe and explain the effect of temperature, pH, and enzyme concentration, on enzyme activity, 4. Define the terms optimum temperature and optimum pH, 5. Recognize the activity of renin enzyme and understand its effect on milk protein.  Enzymes organic compounds protein in nature synthesized in the living cells to accelerate the rate of biological reactions. Factors affecting enzyme activity 1. Effect of substrate concentration 2. Effect of Enzyme Concentration 3. Effect of Co-enzyme Concentration 4. Effect of product Concentration 5. Effect of Temperature 6. Effect of pH 7. Activators and Inhibitors 8. time  Effect of enzyme concentration: The concentration of enzymes influences the rate of a chemical reaction. If enzyme concentration is decreased, then the reaction rate will also decrease. The rate of reaction is directly proportional to the enzyme concentration This rule provided that the substrate is available with sufficient concentration and all the other factors are stable. Substrate exhautio n  Effect of Temperature At 0oC: inactive (reversible) Increase in temperature: increase in activity (For every 10oC increase in temperature there is a double-fold increase in enzymatic activity), till the optimum temperature. 37-45oC (optimum temperature): maximally active. Further increase in temperature: activity is decreased. At 70oC: inactive (irreversible), the enzyme is denaturated All enzymes have the same optimum temperature All the enzymes have the same curve for temperature  Effect of pH Every enzyme has an optimum pH for its maximal activity. Below or above this pH value, the enzyme activity is decreased. At the extremes of pH the enzyme is denatured. Milk composition Milk is almost a complete food because it is rich in all macronutrients ( proteins,fats and carbohydrates) and most of minerals especially calcium and phosphorus. But it is poor in some minerals such as copper and iron. Proteins present in milk are of high biological value as. They include: 1. Caseinogen 60% 2. Lactalbumin, and Lactglobulin (simple soluble proteins) 40%. CASEINOGEN A phosphoprotein Digested by Rennin enzyme (stomach of young calves) parachymosin (in infant, analogue to renin) protein in nature acts optimally at pH 6 and 37oC. In adults this enzyme does not act because of the very low acidity of stomach (pH 1.5-2) which is not suitable for its activity. It is hydrolase enzyme (proteolase) which splits a part of caseinogen polypeptide chain to casein( soluble), which then react with calcium giving calcium caseanate (insoluble) The function of these enzymes is to clot milk and separate it into solid (clot) and liquid (whey). Clotting of the milk is necessary if the milk is to be retained in the stomach long enough for the milk proteins to be digested properly. Young mammals would derive no benefit from the milk they drink if it passed through the stomach too quickly, which is what would happen if it was allowed to remain in its non- clotted state.  Milk clotting ( effect of rennin on milk protein) Milk Clotting takes place in 2 steps: 1. Enzymatic reaction: rennin caseinogen casein (soluble)+small polypeptide 2. Chemical reaction: Ca++ Casein calcium caseanate (insoluble) Calcium caseinate precipitates entangling with it 1. Calcium caseanate 2. milk fat, Milk clot 3. fat soluble vitamins and 4. carotenes. When the clot separates, a fluid is left, formed of 5. H2O, 6. water soluble vitamins 7. lactalbumin, 8. lactglobulin, whe 9. lactose, y 10. Minerals, except Ca (a very small amount). Whey is greenish yellow in color due to its high content of vitamin B2. Experiments : A.Effect of temperature on rennin activity and milk clotting B. Effect of concentration on rennin activity and milk clotting C. Role of calcium in clot formation ? Essential for clot formation ? Ca act as ppt factor and not co-enzyme for rennin Effect of temperature on rennin activity 1. Prepare 3 test tubes each containing 2ml of milk, and then add to them rennin solution as follows: Tube No.1: 2ml Milk + 8 drops of rennin solution. Tube No.2: 2ml Milk + 8 drops of rennin solution. Tube No.3: 2ml Milk + 8 drops of boiled rennin. 2. Put the tubes immediately in the following temperature: o Tube N0.1 at 37oC water bath o Tube No.2 at room temperature o Tube No.3 either at room temp or in the water bath( doesn’t matter as the rennin itself is boiled) 3. Record in minutes the time taken by each to form a firm clot. Results obtained from the previous 3 tubes illustrate the following: Clotting is faster in tube N0.1 in comparison to tube N0.2 (this demonstrates the effect of temperature on the enzyme activity, its is maximal at 37oC). No clotting occurs in tube N0.3 (this demonstrates the effect of boiling on rennin enzyme, it causes its denaturation). Tube N0.3 Tube No. 2 Tube No.1 No clot slower faster  Effect of concentration on rennin activity 1. Prepare 2 test tubes each containing 2ml of milk, and then add to them rennin solution as follows: Tube No.1: 2ml Milk + 4 drops of rennin solution.. 2. Place the tube in a water bath at 37oC 3. Record in minutes the time taken to form a firm clot. 4. Results obtained from the previous 2 experiments illustrate the following:  Clotting is faster in tube 1 in experiment 1 ( has 8 drops of renin at 37oC) in comparison to tube 1 in the 2nd experiment (this demonstrates the effect of concentration of the enzyme on the enzyme activity). Role of calcium in clot formation 1. prepare 2 test tubes, containing the following solutions: 2 tubes containing 2ml Milk + few drops of ammonium oxalate +4 drops of rennin solution. 2. Place the 2 tubes in a water bath at 37oC 3. Observation: o The 2 test tubes do not show any clotting because the Ca of milk in these 2 tubes is completely precipitated as Ca oxalate. This prove that Ca is essential for clot formation  4. boil one of the 2 test tubes, in order to denaturate rennin Then add few drops of CaCl2 solution 5. A clot is formed After boiling And adding Ca This prove that casein is already formed in the absence of Ca, but didn’t ppt till adding calcium i.e. calcium is a ppt factor and not a co- enzyme for rennin Summar y Enzymes are protein that catalyse chemical reactions. Several factors can affect enzyme activity , including temperature , pH and enzyme concentration. The rate of a biochemical reaction increases with rise in temperature and concentration. The main milk protein is caseinogen, it can be precipitated by the renin enzyme.