Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Western blot wikipedia , lookup
Multi-state modeling of biomolecules wikipedia , lookup
P-type ATPase wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Biochemistry wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Biosynthesis wikipedia , lookup
List of types of proteins wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Enzymes Introduction Kinetic reactions: • all chemical reactions in living organisms require enzymes to work For building , breaking down molecules or speed up the reactions. Enzymes are :a biological catalyst. Usually a globular protein molecule produced by living organisms that can speed up a specific chemical reaction without itself being destroyed or changed in any way. They are produced by the living organism and are usually present in only very small amounts in various cells. Enzymes properties Enzymes aren’t used up Enzymes are not changed by the reaction • re-used again for the same reaction with other molecules. Most enzymes are Proteins (tertiary and quaternary structures) Act as Catalyst to accelerates a reaction Are specific for what they will catalyze Enzyme Kinetics Equation The Lock and Key Hypothesis S E E E Enzyme may be used again Enzyme-substrate complex P P Reaction coordinate © 2007 Paul Billiet ODWS Making réactions go faster Increasing the temperature make molecules move faster Biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy. Factors affecting Enzymes substrate concentration pH temperature inhibitors Substrate concentration: Reaction velocity Substrate concentration The increase in velocity is proportional to the substrate concentration © 2007 Paul Billiet ODWS Substrate concentration: Vmax Reaction velocity Substrate concentration Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. Vmax © 2007 Paul Billiet ODWS PH Extreme pH levels will produce denaturation most human enzymes = pH 6-8 depends on where in body pepsin (stomach) = pH 3 trypsin (small intestines) = pH 8 The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it Enzyme activity Trypsin Pepsin 1 3 5 7 pH 9 11 Temperature Effect on rates of enzyme activity Optimum temperature human enzymes 35°- 40°C (body temp = 37°C) Raise temperature (boiling) denature protein = unfold = lose shape Lower temperature T° molecules move slower fewer collisions between enzyme & substrate Temperture and Enzymes ACTIVITY in human Michaelis and menten at low substrate concentrations, the enzyme is not saturated with the substrate and the reaction is not proceeding at maximum velocity whereas when the enzyme is saturated with substrate, maximum velocity is observed. Inhibitors Inhibitors are chemicals that reduce the rate of enzymatic reactions. They are usually specific and they work at low concentrations. They block the enzyme but they do not usually destroy it. Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Reversible inhibitors: There are two categories 1. • • • Competitive: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Come over these problem by adding more substrate Km : The addition of a competitive inhibitor increases the observed Km for a given substrate. Therefore, in the presence of a competitive inhibitor, more substrate is needed to achieve Vmax: Competitive inhibitors do not alter Vmax. The effect of a competitive inhibitor is reversed by increasing [S]. high substrate concentration, the reaction velocity reaches the same Vmax as that observed in the absence of the inhibitor. This is because at the higher concentration the active site will be saturated with substrate which means the inhibitor cannot bind Non-competitive: (allosteric effect) These are not treated by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. Examples Cyanide combines with the Iron in the enzymes cytochrome oxidase. 2. Km : Non-competitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the non-competitive inhibitor. Vmax: Increasing the concentration of substrate does not overcome non-competitive inhibition. Non-competitive inhibitors therefore decrease the Vmax of the reaction. Non-competitive inhibitors therefore simply reduce the amount of active enzyme so they decreaseVmax, but have no effect on Km Kompetitive Inhibition: Km Increases; no change inVmax. Non-kompetitive inhibition: No Km change, but Vmax decreases Michaelis-Menten Equation Glossary Active site: The region of an enzyme molecule which binds the substrate and carries out the catalytic reaction Enzyme : A biological catalyst. Usually a globular protein molecule produced by living organisms that can speed up a specific chemical reaction without itself being destroyed or changed in any way. K m: (Michaelis constant) The substrate concentration at which an enzyme catalysed reaction proceeds at half the maximum velocity. V max: (Maximum velocity) The maximum initial velocity of an enzyme catalysed reaction; determined by increasing the substrate [S] until a constant rate of product formation is achieved (i.e. saturating substrate levels). A CATALYST is anything that speeds up a chemical reaction that is occurring slowly ALKALINE PHOSPHATASE Is a member of the phosphomonoestrases group. Which are highly specific and act an only one substrate, alkaline phosphatase has a broad substrate specificity and is named alkaline phosphatase because its ph optimum is usually around 9 but other broad spectrum phosphoesterases with ph optima less than 7 are termed acid phosphatases. Determination ALP in vitro Alkaline phosphatase activity by following the rate of formation of products. ALP catalyzes in alkaline medium the transfer of the phosphate group from 4 nitrophenylphosphate to 2 amino 2 methyle propanol, librating 4 nitrophenol The catalytic concentration is determined from the rate of 4 nitrophenol formation, measured at 400nm. The 4 nitrophenol is bright yellow but other reactants and products are colorless in aqueous solution. The measurement of ALP activity in vitro is based on artificial substrate p-nitrophenylphosphate. The intensity of yellow color in the reaction solution thus indicates the degree to which enzyme has acted upon substrate.(how much enzyme acted on substrate). Study enzyme kinetics Enzyme kinetics: the study of the rate at which an enzyme works. To examine it, when the substrate available to the enzyme one would do the following: 1. set up the series of tubes containing graded concentrations of substrate 2. At time zero, a fixed amount of the enzyme preparation is added 3. Over the next few minutes, the concentration of product formed, is measured. If the product absorbs light, we can easily do this in a spectrophotometer 4. Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observed is the initial of velocity Vi. 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.) Reagents and instruments 18 labeled plastic tubes Micropipette Spectrophotometer ALP enzyme kit (ready to use) Blood serum 5 N NaOH solution 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. Buffer Substrate Enzyme 5 N NaOH μl μl μl μl 1 100 1800 100 500 2 200 1700 100 500 3 300 1600 100 500 4 400 1500 100 500 5 500 1400 100 500 6 600 1300 100 500 7 700 1200 100 500 8 800 1100 100 500 9 900 1000 100 500 10 1000 900 100 500 11 1100 800 100 500 12 1200 700 100 500 13 1300 600 100 500 14 1400 500 100 500 15 1500 400 100 500 16 1600 300 100 500 17 1700 200 100 500 18 1800 100 100 500 Tube Abs. at 400nm