1、Chapter 2 Enzymes,Reduced DsbA from E. coli,Lysozyme,18:47,Enzyme,2,2.1 Introduction,Definition,History,Bchner, 1897: A breakthrough in Enzymology. Catalysts at work in a living organism could also function completely independently of any life process. Efforts to isolate and purify individual enzyme
2、s were Bchners discoveries. 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.,18:47,Enzyme,3,Characteristics,Three-dimensional structure of the folded protein, determined by the sequence of
3、 the amino acids.,Fragile: mild temperature, pressure, pH, ion strength (ambient conditions).,Lower the activation energy of the reaction, but,18:47,Enzyme,4,Example:,The decomposition of hydrogen peroxide(H2O2):,From Arrhenius equation:,We have,18:47,Enzyme,5,The interaction between the enzyme and
4、its substrateweak force: van der Waals forces Hydrogen bonding,Specific: Catalyze one kind of reaction Involving certain substrates,Need for cofactors or coenzyme: Cofactors: metal ions:Mg, Zn, Mn, Fe Coenzyme: a complex organic molecule:NAD, FAD, CoA, some vitamins,18:47,Enzyme,6,Enzymes are named
5、by adding the suffix ase to the: End of the substrate Such as urease The reaction catalyzed Such as alcohol dehydrogenase,Nomenclature,Enzymes using familiar names: Pepsin in the digestive tract Trypsin in the digestive tract Rennin used in cheese making “Old yellow”, which caused browning of sliced
6、 apples,18:47,Enzyme,7,EC (Enzyme Commission)SIX classes numbered in FOUR digits,Classification,The first digitalThe second digital The third digital The fourth digital, main classes, actual substance,18:47,Enzyme,8,Oxidoreductases,First digit 1 the class oxidoreductases. Second digit the donor of h
7、ydrogen atom or electron involved. Alcohol Aldehyde or ketone Alkene CH=CH- Primary amine Secondary amine NADH, NADPH Third digit hydrogen atom or electron acceptor. NAD+, NADP+ Fe3+ O2 Otherwise unclassified Fourth digit number for further identification.,18:47,Enzyme,9,Transferases,First digit 2 t
8、he class transferases. Second digit general type of groups transferred. 1-carbon group Aldehyde or ketone Acyl group(-CO-R-) Glycosyl group Phosphate group Sulphur containing group Third digit provide details on the exact name of the group transferred.,Transferases catalyze the functional group tran
9、sfer reactions, with a general form given below:AX + B BX + A,18:47,Enzyme,10,Hydrolases,First digit 3 the class hydrolases. Second digit the type of bond hydrolyzed Ester Glycosidic Peptide Other C-N bonds Acid anhydrides,Hydrolyases catalyze hydrolytic reactions, with a general form given below:A-
10、X + H2O X-OH + HA,18:47,Enzyme,11,Lyases,First digit 4 the class lyases. Second digit the type of binds broken. C-C C-O C-N C-S Third digit The group removed. Carboxyl Aldehyde Keto acid Fourth digit number for further identification.,Lyases catalyze the non-hydrolytic removal of groups from substan
11、ces. Often the product contains a double bond.,18:47,Enzyme,12,Isomerases,First digit 5 the class isomerases. Second digit the type of reaction involved. Racemization or epimerization Cis-trans isomerization Intramolecular oxidoreductases Intramolecular transfer reactions Third digit the type of mol
12、ecule undergoing isomerization. Amino acids Hydroxyacids carbohydrates Fourth digit number for further identification.,18:47,Enzyme,13,Ligases,First digit 6 the class ligases. Second digit the type of bonds formed. C-O C-S C-N C-C,Ligases catalyze the synthesis of various types of bonds, where the r
13、eactions are coupled with breakdown of energy-containing materials, such as ATP or nucleoside triphosphates. X + Y + ATP X-Y + ADP + PiX + Y + ATP X-Y + AMP + PPi,18:47,Enzyme,14,Some Examples of Enzyme,Alcohol Dehydrogenase EC 1.1.1.1 Glucose Oxidase EC 1.1.3.4 Catalase EC 1.11.1.6 Tryptophan 2,3-d
14、ioxygenase EC 1.13.11.11 Pyruvate Kinase EC 2.7.1.40 Creatine Kinase EC 2.7.3.2 Alpha-amylase EC 3.2.1.1 Chitinase EC 3.2.1.14 Oxaloacetate Decarboxylase EC 4.1.1.3 Lactate Racemase EC 5.1.2.1 Ribose Isomerase EC 5.3.1.20 AcetateCoA Ligase EC 6.2.1.1 Glutathione Synthase EC 6.3.2.3,18:47,Enzyme,15,2
15、.2 How Enzymes Work?,Lock and Key ModelDeveloped by Emil Fischer in 1895.The enzymes and substrates combine because they have complementary molecular geometries.,18:47,Enzyme,16,An Example of Lock-Key Model,18:47,Enzyme,17,Induced-Fit Model Like a hand and glove.The enzyme is a molecule whose confor
16、mation can change as the substrate approaches and starts to bind.,18:47,Enzyme,18,An Example of Induced-Fit Model,18:47,Enzyme,19,2.3 Enzyme Kinetics,Two assumptions:,Reaction occurred in well-mixed reactor.That is to say, spatially uniform. Only initial rate is used:,which has the unit of M/s.,18:4
17、7,Enzyme,20,2.3.1 Mechanistic Models for Simple Enzyme Kinetics,Single-substrate kinetics was first developed: V.C.R. Henri in 1902 L. Michaelis and M.L. Menten in 1913,A simple reaction scheme:,Saturation kinetics can be obtained for the reaction scheme above.,18:47,Enzyme,21,The same few initial s
18、teps in deriving a rate expression:,18:47,Enzyme,22,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.,18:47,Enzyme,23,A low value of suggests that the enzyme has a hig
19、h affinity for the substrate.,18:47,Enzyme,24,Experimental data demonstrated the concentration profiles.,ASSUMPTION: Initial substrate concentration greatly exceeds the initial enzyme concentration.,is small, then,18:47,Enzyme,25,18:47,Enzyme,26,Both Give Saturation Kinetics,Saturation kinetics, sim
20、ilar to Langmuir-Hinshelwood isothermal adsorption kinetics, which shows a first-order kinetics at the low substrate concentrations, but zero-order kinetics at high substrate concentrations.,18:47,Enzyme,27,Questions,Why only initial rate can be used ?,Why a low value of suggests that theenzyme has
21、a high affinity for the substrate ?,18:47,Enzyme,28,2.3.2 Experimentally Determining Rate Parameters for Michaelis-Menten Type Kinetics,With known S0 and E0, we can calculate the initial rate:,18:47,Enzyme,29,Double-Reciprocal Plot (Lineweaver-Burk Plot),By rearrangement, we can get,18:47,Enzyme,30,
22、Eadie-Hofstee Plot,By rearrangement, we can also get,18:47,Enzyme,31,Hanes-Woolf Plot,By rearrangement, we can also get,18:47,Enzyme,32,Batch Kinetics,The time course of variation of S in a batch enzymatic reaction can be determined from,By integration to yield,or,A plot of versus results in a line ofslope and intercept of .,
