生物反应工程chap2enzyme1ppt课件

Chapter 2 Enzymes Reduced DsbA from E. coliLysozyme * 1Enzyme 2.1 Introduction Definition History u Bchner, 1897: A breakthrough in Enzymology. l Catalysts at work in a living organism could also function completely independently of any life process. l Efforts to isolate and purify individual enzymes were Bchners discoveries. u Sumner, 1926: First isolation of a pure enzyme. Enzymes are usually proteins of high molecular weight (15,000 MW several million Daltons) that act as catalysts. Date 2Enzyme Characteristics u Three-dimensional structure of the folded protein, determined by the sequence of the amino acids. u Fragile: mild temperature, pressure, pH, ion strength (ambient conditions). u Lower the activation energy of the reaction, but Doesnt affect Free-energy change Equilibrium constant Date 3Enzyme Example: The decomposition of hydrogen peroxide(H2O2): 18kcal/mol Uncatalyzed reaction at 20 Colloidal platinum Chemically catalyzed Hydrogen peroxidase Enzymatically catalyzed 13kcal/mol 7kcal/mol From Arrhenius equation: We have, Date 4Enzyme u The interaction between the enzyme and its substrateweak force: l van der Waals forces l Hydrogen bonding u Specific: l Catalyze one kind of reaction l Involving certain substrates u Need for cofactors or coenzyme: l Cofactors: metal ions: Mg, Zn, Mn, Fe l Coenzyme: a complex organic molecule: NAD, FAD, CoA, some vitamins Date 5Enzyme u Enzymes are named by adding the suffix ase to the: l End of the substrate Such as urease l The reaction catalyzed Such as alcohol dehydrogenase Nomenclature u Enzymes using familiar names: l Pepsin in the digestive tract l Trypsin in the digestive tract l Rennin used in cheese making l “Old yellow”, which caused browning of sliced apples Date 6Enzyme u EC (Enzyme Commission) u SIX classes numbered in FOUR digits Classification The first digital The second digital The third digital The fourth digital Type of reaction catalyzed main classes actual substance Date 7Enzyme u Oxidoreductases l First digit 1 the class oxidoreductases. l Second digit the donor of hydrogen atom or electron involved. 1. Alcohol 2. Aldehyde or ketone 3. Alkene CH=CH- 4. Primary amine 5. Secondary amine 6. NADH, NADPH l Third digit hydrogen atom or electron acceptor. 1. NAD+, NADP+ 2. Fe3+ 3. O2 4. Otherwise unclassified l Fourth digit number for further identification. Date 8Enzyme u Transferases l First digit 2 the class transferases. l Second digit general type of groups transferred. 1. 1-carbon group 2. Aldehyde or ketone 3. Acyl group(-CO-R-) 4. Glycosyl group 7. Phosphate group 8. Sulphur containing group l Third digit provide details on the exact name of the group transferred. Transferases catalyze the functional group transfer reactions, with a general form given below: AX + B BX + A Date 9Enzyme u Hydrolases l First digit 3 the class hydrolases. l Second digit the type of bond hydrolyzed 1. Ester 2. Glycosidic 4. Peptide 5. Other C-N bonds 6. Acid anhydrides Hydrolyases catalyze hydrolytic reactions, with a general form given below: A-X + H2O X-OH + HA Date 10Enzyme u Lyases l First digit 4 the class lyases. l Second digit the type of binds broken. 1. C-C 2. C-O 3. C-N 4. C-S l Third digit The group removed. 1. Carboxyl 2. Aldehyde 3. Keto acid l Fourth digit number for further identification. Lyases catalyze the non-hydrolytic removal of groups from substances. Often the product contains a double bond. Date 11Enzyme u Isomerases l First digit 5 the class isomerases. l Second digit the type of reaction involved. 1. Racemization or epimerization 2. Cis-trans isomerization 3. Intramolecular oxidoreductases 4. Intramolecular transfer reactions l Third digit the type of molecule undergoing isomerization. 1. Amino acids 2. Hydroxyacids 3. carbohydrates l Fourth digit number for further identification. Date 12Enzyme u Ligases l First digit 6 the class ligases. l Second digit the type of bonds formed. 1. C-O 2. C-S 3. C-N 4. C-C Ligases catalyze the synthesis of various types of bonds, where the reactions are coupled with breakdown of energy -containing materials, such as ATP or nucleoside triphosphates. X + Y + ATP X-Y + ADP + Pi X + Y + ATP X-Y + AMP + PPi Date 13Enzyme uSome Examples of Enzyme l Alcohol Dehydrogenase EC 1.1.1.1 l Glucose Oxidase EC 1.1.3.4 l Catalase EC 1.11.1.6 l Tryptophan 2,3-dioxygenase EC 1.13.11.11 l Pyruvate Kinase EC 2.7.1.40 l Creatine Kinase EC 2.7.3.2 l Alpha-amylase EC 3.2.1.1 l Chitinase EC 3.2.1.14 l Oxaloacetate Decarboxylase EC 4.1.1.3 l Lactate Racemase EC 5.1.2.1 l Ribose Isomerase EC 5.3.1.20 l AcetateCoA Ligase EC 6.2.1.1 l Glutathione Synthase EC 6.3.2.3 Date 14Enzyme 2.2 How Enzymes Work? Lock and Key Model Developed by Emil Fischer in 1895. The enzymes and substrates combine because they have complementary molecular geometries. Date 15Enzyme An Example of Lock-Key Model Date 16Enzyme Induced-Fit Model Like a hand and glove. The enzyme is a molecule whose conformation can change as the substrate approaches and starts to bind. Date 17Enzyme An Example of Induced-Fit Model Date 18Enzyme 2.3 Enzyme Kinetics Two assumptions: a. Reaction occurred in well-mixed reactor. That is to say, spatially uniform. b. Only initial rate is used: Two major approaches: a. Rapid equilibrium approach; b. Quasi-steady-state approach. which has the unit of M/s. Date 19Enzyme 2.3.1 Mechanistic Models for Simple Enzyme Kinetics Single-substrate kinetics was first developed: l V.C.R. Henri in 1902 l L. Michaelis and M.L. Menten in 1913 A simple reaction scheme: Saturation kinetics can be obtained for the reaction scheme above. Date 20Enzyme The same few initial steps in deriving a rate expression: The rate of variation of the ES complex: Eq. (2.2) The conservation equation on the enzyme: Eq. (2.3) The rate of product formation: Eq. (2.1) Date 21Enzyme Rapid Equilibrium Assumption (Developed by Henri and Michaelis and Menten) ASSUMPTION: A rapid equilibrium between the enzyme and the substrate can be achieved to form an ES complex. The dissociation constant: Eq. (2.4) Substituting Eq. (2.3) into Eq. (2.4) gives, Eq. (2.5) Date 22Enzyme Substituting Eq. (2.5) into Eq. (2.1) yields, Eq. (2.6) where , . A low value of suggests that the enzyme has a high affinity for the substrate. the maximum forward velocity of the reaction; if the amount of the enzyme changes, changes the Michaelis-Menten constant Date 23Enzyme Experimental data demonstrated the concentration profiles. ASSUMPTION: Initial substrate concentration greatly exceeds the initial enzyme concentration. is small, then The Quasi-Steady-State Assumption (Developed by G.E. Briggs and J.B.S. Haldane) Date 24Enzyme From Eq. (3.2) and the assumption, we have, Eq. (2.7) Substituting Eq. (2.3) in to Eq. (2.7) gives Eq. (2.8) Substituting Eq. (2.8) in to Eq. (2.1) yields Eq. (2.9) where , . Date 25Enzyme Both Give Saturation Kinetics Saturation kinetics, similar to Langmuir-Hinshelwood isothermal adsorption kinetics, which shows a first- order kinetics at the low substrate concentrations, but zero-order kinetics