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Outline of Enzyme Introduction Enzyme kinetics - Mechanistic models for simple enzyme kinetics - Effect of pH and Temperature Immobilized Enzyme System - Method of immobilization - Diffusional limitations - Electrostatic and steric effects Large-scale production and application of enzyme. Slide 2 What is Enzyme? An enzyme is a protein molecule that is a biological catalyst with the following characteristics. Effective to increase the rate of a reaction. Most cellular reactions occur about a million times faster than they would in the absence of an enzyme. Specifically act with one reactant (called a substrate) to produce products. maltase that converts maltose to glucose Be regulated from a state of low activity to high activity and vice versa. e.g. some enzyme activity is inhibited by the product. Be versatile: More than 3000 enzymes are identified Slide 3 Enzyme Enzyme has high molecule weight (15,000< mw< several million Daltons). Holoenzyme is an enzyme contains non-protein group. Such non-protein group is called cofactor such as metal ions, Mg, Zn, Mn, Fe Or coenzyme, such as a complex organic molecule, NAD, or vitamins. Apoenzyme is the protein part of holoenzyme. Holoenzyme = apoenzyme + cofactor (coenzyme) Slide 4 Enzyme nomenclature Enzyme is named by adding the suffix ase - to the end of the substrate that is to be converted to the desired product. e.g. urease that changes urea into ammonium carbonate. protease that converts protein or polypeptides to smaller molecules such as amino acids. - to the reaction catalyzed. e.g. phosphoglucose isomerase that converts glucose-6- phosphate to fructose-6-phosphate. Alcohol dehydrogenase that catalyzes the removal of hydrogen from alcohol. Slide 5 Enzyme classification International Classification of Enzymes by the International Classification Commission in 1864. Enzymes are substrate specific and are classified according to the reaction they catalyze. Enzyme Nomenclature, 1992, Academic Press, San Diego, California, ISBN 0-12-227164-5. http://www.chem.qmul.ac.uk/iubmb/enzyme/ Slide 6 Enzymes can be classified into six main classes: - Oxidoreductases: catalyze the oxidation and reduction e.g. CH 3 CH 2 OH CH 3 CHO+H + - Transferases: catalyze the transfer of a functional group (e.g. a methyl or phosphate group) from one molecule (called the donor) to another (called the acceptor). AX + B A + BX - Hydrolases:catalyze the hydrolysis of a chemical bond. AB + H 2 O AOH + BH, e.g. peptide bond Enzyme classification Slide 7 - Lyases:catalyze the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure e.g. CH 3 COCO-OH CH 3 COCHO (dehydratase) - Isomerases:catalyse the interconversion of isomers. e.g.phosphoglucose isomerase that converts glucose-6- phosphate to fructose-6-phosphate. - Ligases:catalyse the joining of two molecules by forming a new chemical bond, with accompanying hydrolysis of ATP or other similar molecules ATP + L-tyrosine + tRNATyr = AMP + diphosphate + L-tyrosyl-tRNATyr Enzyme classification Slide 8 Each main class contains subclasses, subsubclasses and subsubsubclasses. There are four levels of classes in total. e.g. Reaction: CH 2 CH 2 OH+NAD + CH 3 CHO (acetaldehye)+NADH+H + Systematic name: alcohol NAD oxidoreductase (22.214.171.124) Trivial name: alcohol dehydrogenase 1. Oxidation-reduction reaction 1.1 Acting on R-OH group of substrates 1.1.1 Requires NAD + or NADP + as hydrogen acceptor 126.96.36.199 Specific substrate is ethyl alcohol Slide 9 Mechanism of enzyme catalysis What is a catalyst? A catalyst is a substance that accelerates the rate (speed) of a chemical reaction without itself being consumed or transformed. It participates in reactions but is neither a chemical reactant nor chemical product. S + C (catalyst)P + C (catalyst) SP Slide 10 Mechanism of enzyme catalysis Catalysts provide an alternative pathway of lower activation energy for a reaction to proceed whilst remaining chemically unchanged themselves. Free energy change Slide 11 Catalysts lower the activation energy of the reaction catalyzed by binding the substrate and forming an catalyst-substrate complex which produces the desired product. Catalysts lower the activation energy of the catalyzed reaction, but does not affect free energy change or equilibrium constant. Mechanism of enzyme catalysis Slide 12 The reaction rate v is strongly affected by the activation energy of the reaction. v = k*f(S) -f(S) denotes the function of substrate concentration -k is the rate constant which can be expressed by Arrhenius equation ( H.S. Fogler, Chemical Reaction Engineerng, Prentice-Hall Inc., 2005) k=A*exp(-Ea/R*T) A is a constant for a specific system, Ea is the activation energy, R is the universal gas constant, and T is the temperature (in degrees Kelvin). When Ea is lowered, k is increased, and so is the rate. Mechanism of enzyme catalysis Slide 13 Catalysts do not affect free energy change or equilibrium constant of the catalyzed reaction. Free energy (G) is the energy stored in the bonds of a chemical that can be harnesses to do work. Free energy change (G) of a reaction refers to the change between the free energy in the product (s) and that in the substrate(s). Mechanism of enzyme catalysis Slide 14 For uncatalyzed reaction, free energy change G, uncatalyzed =G(P)-G(S) For catalyzed reaction, free energy change G, catalyzed =G(P)-G(S) Therefore, G, uncatalyzed = G, catalyzed S + C (catalyst)P + C (catalyst) SP For an example, Slide 15 Mechanism of enzyme catalysis Free energy change determines the reaction equilibrium the maximum amounts of the product could be theoretically produced. Reaction equilibrium is represented by reaction equilibrium constant K eq,= p [P]/ s [S] - G, uncatalyzed =RTln K eq [ ] represents the concentration of the compounds. p and s are activity constants of the product and the substrate, respectively. S + C (catalyst)P + C (catalyst) SP Slide 16 Mechanism of enzyme catalysis Catalysts can not increase the amounts of the product at reaction equilibrium. Catalysts can only accelerate the reaction rate to reach the reaction equilibrium. Slide 17 Mechanism of enzyme catalysis Enzymes are catalysts and have the characteristics of common catalysts. Particularly, Enzymes are - Efficient - Specific - regulated - versatile Slide 18 Efficiency of Enzyme Catalysis For an example, in the reaction of decomposition of hydrogen peroxide, the activation energy Ea,o of the uncatalyzed reaction at 20 o C is 18 kcal/mol, whereas that for chemically catalyzed (Pt) and enzymatically catalyzed (catalase) decomposition are 13 kcal/mol (Ea,c) and 7 kcal/mol (Ea, en), respectively. Compare the reaction rates at these three different conditions. Slide 19 The forward reaction rate r (moles/L-s), r = k*f(H 2 O 2 ) k is the forward reaction rate constant, f(H 2 O 2 ) is a function of substrate perioxide concentration. Efficiency of Enzyme Catalysis 2H 2 O 2 2H 2 O + O 2 Slide 20 If k is represented by k 0, k c, and k en, respectively, for the uncatalyzed, chemically catalyzed (Pt) and enzymatically catalyzed reactions, the corresponding reaction rates ( r 0, r c, r en ) can be expressed as follows r 0 = k 0 *f(H 2 O 2 ) r c = k c *f(H 2 O 2 ) r en = k en *f(H 2 O 2 ) Slide 21 The ratio of the chemically catalyzed rate to the uncatalyzed rate can be calculated by as follows: r c /r 0 = k c *f(H 2 O 2 )/k 0 *f(H 2 O 2 ) Similarly, The ratio of the enzymatically catalyzed rate to the uncatalyzed rate can be calculated by as follows: r en /r 0 = k en *f(H 2 O 2 )/k 0 *f(H 2 O 2 ) At 20 o C, with the same initial concentration of reactant hydrogen peroxide, f(H 2 O 2 ) remains the same for the above three process at initial conditions, the initial reaction rates vary with the rate constants. r c /r 0 = k c /k 0 r en /r 0 = k en /k 0 Slide 22 Using Arrhenius Equations yields, r c /r 0 = k c /k 0 = (A*exp(-Ea,c/R*T))/(A*exp(-Ea,o/R*T)) r en /r 0 = k en /k 0 = (A*exp(-Ea,en/R*T))/(A*exp(-Ea,o/R*T)) Where Ea,o, Ea,c and Ea,en are activation energy for uncatalyzed reaction, chemically catalyzed and enzymatically catalyzed reactions, respectively. A is a constant and remains same for the specific system, the rate ratios could be simplified to: r c /r 0 = (exp(-Ea,c/R*T))/(exp(-Ea,o/R*T)) r en /r 0 = (exp(-Ea,en/R*T))/(exp(-Ea,o/R*T)) Slide 23 When T= 20 o C = 273+20 (K) = 293 K, Ea,o = 18 kcal/mol, Ea,c = 13 kcal/mol (Ea,c) and Ea, en =7 kcal/mol, respectively. R = 8.314 J mol -1 K -1, 1 cal = 4.18 J. Then substituting the parameters in the rate ratio eqns. with these values, yields, r c /r 0 = (exp(-Ea,c/R*T))/(exp(-Ea,o/R*T)) = (exp(-13000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293)) =? r en /r 0 = (exp(-Ea,en/R*T))/(exp(-Ea,o/R*T)) =(exp(-7000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293) =? Slide 24 When T= 20 o C = 273+20 (K) = 293 K, Ea,o = 18 kcal/mol, Ea,c = 13 kcal/mol (Ea,c) and Ea, en =7 kcal/mol, respectively. R = 8.314 J mol -1 K -1, 1 cal = 4.18 J. Then substituting the parameters in the rate ratio eqns. with these values, yields, r c /r 0 = (exp(-Ea,c/R*T))/(exp(-Ea,o/R*T)) = (exp(-13000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293)) =5.1X10 3 r en /r 0 = (exp(-Ea,en/R*T))/(exp(-Ea,o/R*T)) =(exp(-7000*4.18/8.314*293))/(exp(-18000*4.18/8.314*293) =1.4X10 8 Slide 25 Enzyme catalysis is efficient! - If it takes 1 hr to complete the reaction with enzyme - it needs to take 1.4X10 8 hrs = 5833333 days = 15981years to complete the same reaction without enzyme catalysis. Slide 26 Specificity of Enzyme Catalysis Much of the catalytic power of enzymes comes from their bringing substrates together in favorable orientations to promote the formation of the transition states in enzyme- substrate (ES) complexes. The substrates are bound to a specific region of the enzyme called the active site. Most enzymes are highly selective in the substrates that the