生化教學(xué)課件

1、Chapter 5 Enzymology (酶學(xué)),Physiological significance of enzymes Composition, structure and properties of enzyme Description of enzyme kinetics and inhibition Chemical mechanisms of catalysis Regulation of enzyme activities,Key topics,Key words,Coenzyme (輔酶) Prosthetic group (輔基) Substrate (底物) Catal

2、yze (催化) Transition state (過渡態(tài)) Steady state (穩(wěn)態(tài)) Kinetics (動(dòng)力學(xué)) Initial velocity (Initial rate) (V0, 初速度) Maximum velocity (Vmax, 最大速度) Michaelis constant (米氏常數(shù)),Ribozyme (核酶) Abzyme (抗體酶) Isozyme (同工酶) Noncompetitive inhibition (非競(jìng)爭(zhēng)性抑制作用) Uncompetitive inhibition (反競(jìng)爭(zhēng)性抑制作用),What are enzymes?,Enzym

3、es are catalytically active biological macromolecules Most enzymes are globular proteins, however some RNAs also catalyze reactions Study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s Study of enzymes has dominated biochemistry in the past and continues to do

4、so,(1). Much of the early history of biochemistry is the history of enzyme research (2). Biological catalysts were first recognized in studying animal food digestion and sugar fermentation with yeast (brewing and wine making) (3). Ferments (i.e., enzymes, meaning in “in yeast”) were thought (wrongly

5、) to be inseparable from living yeast cells for quite some time (Louis Pasteur),5.1 Introduction to enzymes,(4). Yeast extracts were found to be able to ferment sugar to alcohol (Eduard Buchner, 1897, who won the Nobel Prize in Chemistry in 1907 for this discovery) (5). Enzymes were found to be prot

6、eins (1920s to 1930s, James Sumner on urease and catalase (過氧化氫酶), “all enzymes are proteins”, John Northrop on pepsin and trypsin, both shared the 1946 Nobel Prize in Chemistry) (6). Almost every chemical reaction in a cell is catalyzed by an enzyme (thousands have been purified and studied, many m

7、ore are still to be discovered!) (7). Catalytic RNA were found in the 1980s (Sidney Altman and Thomas Cech, Nobel Prize in Chemistry in 1989),The Nobel Prize in Chemistry 1989,for their discovery of catalytic properties of RNA,Sidney Altman,Thomas R. Cech,Canada and USA,Yale University New Haven, CT

8、, USA,b. 1939,USA,University of Colorado Boulder, CO, USA,b. 1947,Sidney Altman 1989 Nobel Laureate in Chemistry,(8). Abzyme (抗體酶)亦被稱為Catalytic antibody (催化性抗體),Antibody: 與抗原特異結(jié)合的免疫球蛋白,Abzyme (from antibody enzyme): 指具有催化功能的抗體分子，在抗體分子的可變區(qū)(即肽鏈的N端)是識(shí)別抗原的活性區(qū)域，這部分區(qū)域被賦予了酶的屬性,1986年美國(guó)Schultz和Lerner兩個(gè)實(shí)驗(yàn)室同時(shí)在

9、Science上發(fā)表論文，報(bào)道他們成功地得到了具有催化活性的抗體,What is the difference between an enzyme and a protein?,Protein,All enzymes are proteins except some RNAs and not all proteins are enzymes,RNA,Enzymes,5.2 Definition of enzyme,Enzymes are biological catalysts. A Catalyst is defined as a substance that increases the r

10、ate of a chemical reaction without being itself changed in the process.” The protein tertiary structure provides a defined region optimized for the acceleration of chemical transformations (Active site).,5.3 Properties of enzymes,Catalyst - speeds up attainment of reaction equilibrium. Enzymatic rea

11、ctions - 103 to 1017 faster than the corresponding uncatalyzed reactions. Mild reaction conditions- 37, physiological pH, ambient atmospheric pressure. Substrates - highly specific reactants for enzymes.,Stereospecificity (立體特異性)- many enzymes act upon only one stereoisomer of a substrate Reaction s

12、pecificity - enzyme product yields are essentially 100% (there is no formation of wasteful byproducts) Active site - where enzyme reactions take place Enzymatic reactions are often regulated, especially those associated with key metabolic transformations,Substrate specificity,5.4 Chemical compositio

13、n of enzymes,(1). Simple protein (單純蛋白質(zhì))酶類,(2). Conjugated protein (綴合蛋白質(zhì))酶類,Holoenzyme(全酶) = Apoenzyme(酶蛋白) + Cofactor (輔助因子) (輔酶、輔基或金屬離子),Coenzyme (輔酶): loosely bound to enzyme,Prosthetic group (輔基): very tightly or even covalently bound to enzyme,5.5 Classification of enzymes,1). Monomeric enzyme

14、 (單體酶): 大多數(shù)水解酶類,2). Oligomeric enzyme (寡聚酶): 偶數(shù)亞基,3). Multienzyme complex (多酶復(fù)合物): 幾種酶通過非共價(jià)鍵彼此嵌合。 如Fatty acid synthase (脂肪酸合酶),(1). By their composition,Nomenclature of enzyme,Many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to a word or phrase describing th

15、eir activity (type of reaction). e.g. Urease (脲酶) DNA polymerase (DNA聚合酶).,(2). International classification -By the reactions they catalyze (Six classes): 氧,轉(zhuǎn),水, 裂,異,連,1). Oxidoreductases (dehydrogenases)氧化還原酶類和脫氫酶類 Catalyze oxidation-reduction reactions,2). Transferases(轉(zhuǎn)移酶類),Catalyze group transf

16、er reactions,3). Hydrolases (水解酶類),Catalyze hydrolysis reactions where water is the acceptor of the transferred group,4). Lyases, Synthases (裂合酶類),Lyases catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination (Synthases catalyze the addition to a double

17、bond, the reverse reaction of a lyase),5). Isomerases (異構(gòu)酶類),Catalyze isomerization reactions,6). Ligases,連接酶類(synthetases, 合成酶),Catalyze ligation, or joining of two substrates Require chemical energy (e.g. ATP),Each enzyme is given a systematic name and a unique 4-digit identification number for id

18、entification by the Enzyme Commission (E.C.) of IUBMB (since 1964),Lactate dehydrogenase (lactate:NAD+ oxidoreductase),lactate + NAD+ pyruvate + NADH + H+,1,Indicates type of substrate,Indicates type of cofactor,5.6 Isolation, purification and activity determination of enzyme,(1). Procedure of isola

19、tion and purification,1). Material selection,2). Cell breaking,3). Extraction,4). Nucleic acid and saccharide removal,5). Isolation and purification,7). Storage,6). Crystallization(結(jié)晶),Note in isolation and purification,-All steps at 04,-Avoiding vigorous stirring,-Addition of protection reagent e.g

20、. EDTA and 少量-巰基乙醇,-在分離提純過程中要不斷測(cè)定酶活力和蛋白質(zhì)濃度，從而求得比活力，還要計(jì)算總活力,Three measures of enzyme activity are commonly used: Turnover number (轉(zhuǎn)換數(shù)，kcat) - number of substrate molecules turned over per enzyme molecule per second International unit (國(guó)際單位，IU) - amount of enzyme that produces 1 mol of product per min

21、ute. i.e. 1 IU = 1 mol /min Specific activity(比活力) - IU/mg protein,(2) Definitions of enzyme activity,(3). Methods to determine the activity of enzyme,1). Spectrophotometry (分光光度法),該法要求酶的底物和產(chǎn)物在紫外或可見光部分光吸收不同,e.g. Lactate dehydrogenase (乳酸脫氫酶，LDH),L-乳酸 + NAD+ 丙酮酸 + NADH + H+,LDH,2). Fluorescence (熒光法)

22、,3). Enzyme coupling assay (酶偶聯(lián)分析法),4). Electrochemical method (電化學(xué)法),5.7 Enzyme kinetics (酶動(dòng)力學(xué)),Why study enzyme kinetics?,Enzyme kinetics provides a measure of the maximum catalytic rate of an enzyme and its affinity for substrates and inhibitors. Additionally combined with structural and protein

23、chemistry studies, enzyme kinetics provides a powerful viewport into the enzymes mechanism of catalysis.,Enzymes lower the energy of activation G. Both forward and reverse rates are enhanced but equilibrium constant (平衡常數(shù)) is unchanged,Transition state: the point of highest energy,Reaction coordinat

24、e comparing enzyme-catalyzed and uncatalyzed reactions,E + S ES EP E + P,An imaginary enzyme (stickase) designed to catalyze breakage of a metal stick,(1). Substrate concentration (S) affects the catalytic velocity (rate),1). Initial velocity (rate, V0),S is a key factor affecting the rate. Studying

25、 the effects of S is complicated for S changes during the course of reaction as S is converted to P. One simplifying approach in kinetics experiments is to measure the V0,Initial velocities of enzyme-catalyzed reactions,Tangent (切線),Effect of S on the V0 of an enzyme-catalyzed reaction Rectangular h

26、yperbola(矩形雙曲線),At high S the reaction rate is independent of S (zero order) At low S reaction is first order,Vmax is reached when an enzyme is saturated with S (high S),When S E, every enzyme binds a molecule of substrate (E is saturated with S ) Under these conditions the V depends only upon E,ES

27、complex formed when specific substrates fit into the enzyme active site (at the beginning of reaction),2). Quantitative expression of relationship between S and V0,Michaelis-Menten equation,Equation describes V vs S plots KM : Michaelis constant,Note: here V means V0,Leonor Michaelis and Maud Menten

28、 deduced the equation based on the exist of intermediate (ES) in the enzyme reaction in 1913,Derivation of Michaelis-Menten equation based on intermediate (ES, 中間復(fù)合物),Leonor Michaelis (1875-1949),Maud Menten (1879-1960),E + S ES E + P,k1,k3,k2,At the beginning of reaction, dissociation of ES to form

29、 P is assumed to be irreversible, an assumption under initial velocity conditions,V = dP / dt = k3ES Rate of formation of ES = k1ES Rate of breakdown of ES = (k2 + k3)ES,Briggs and Haldane (1925) proposed that after the initial stage of reaction, ES complex (“Michaelis”) remains constant until S is

30、depleted ie. dES / dt = 0 - Steady state (穩(wěn)態(tài)),For steady state k1ES = (k2 + k3)ES and rearranging,The ratio of kinetic constants is defined as the Michaelis constant, Km Km = (k2 + k3)/k1,Substitution of Km yields ES = ES/Km,Since we assume that the uncombined S E, and the total E (ET ) is fixed, th

31、en ES can be written in terms of total E and S. E = ET - ES or ES = (ET - ES)S/Km,Simplify the expression for ES,And substitution into the velocity equation,When S Km, then Vmax = k3ET, or,Note: V means V0,When SKm , the Michaelis-Menten equation simplifies to V = Vmax; that is, the V is independent

32、 of S. When KmS, then V = Vmax S/Km or V is linearly dependent on S.,In summary The equation is derived from Steady-state conditions: Rate of ES formation = Rate of ES decomposition Michaelis constant: Km = (k2 + k3) / k1 Vo depends upon rate of conversion of ES to E + P Vo = k3ES,3). Meanings of Km

33、,Km = S when V = 1/2 Vmax , so, the unit of Km is as S,For a specific substrate Km is a constant for the enzyme (measured under defined experimental conditions, eg. pH, ionic strength, etc) The lower the value of Km, the tighter the substrate binding Km can be a measure of the affinity of E for S,Km

34、 often near physiological S. The ratio S/KM typically ranges from 0.01 to 1 with Km ranging from 10-8 to 10-1 M,Km values for enzymes are typically just above S, so that the enzyme rate is sensitive to small changes in S,4). Some other important parameters of enzyme,Catalytic constants (kcat ，催化常數(shù)),

35、 also called turnover number kcat - first order rate constant for conversion of ES complex to E + P. kcat most easily measured when the E is saturated with S (region A) Turnover number: represents the number of moles of substrate converted to product per second per mole of enzyme. The unit for kcat

36、and Turnover number is s-1,kcat / Km is a second order rate constant with units of M-1s-1 for E + S E + P at low S concentrations (region B),Meanings of kcat and kcat/ Km,kcat / Km is also called specificity constant (特異性常數(shù)),The best way to compare the catalytic efficiencies of different enzymes or

37、the turnover of different substrates by the same enzyme is to compare the ratio kcat/Km for the two reactions,kcat/Km is a measure of catalytic efficiency,只有Kcat/KM可以客觀地比較不同的酶或同一種酶催化不同底物的催化效率,5). Measurement of Km and Vmax,(i) Plot of V vs S,缺點(diǎn): 不準(zhǔn)，因?yàn)椋?(ii) The double-reciprocal (雙曲線) -Lineweaver-Bu

38、rk plot,Transform the Michaelis-Menten equation to,The double-reciprocal Lineweaver-Burk plot is a linear transformation of the Michaelis-Menten plot (1/V vs 1/S),(iii) Plot of V vs (Eadie-Hofstee plot),(3). Kinetics of bisubstrate (雙底物) and biproduct (雙產(chǎn)物) reactions,A + BP + Q,sequential reactions

39、(序列反應(yīng)),ping pong reactions (乒乓反應(yīng) ),orderd reaction (有序反應(yīng)),random reactions (隨機(jī)反應(yīng) ),Sequential reactions: involving a ternary complex.,Ping pong reactions: no ternary complex is formed.,ATP + Glucose ADP + Glucose-6-phosphate, A與其產(chǎn)物Q相互競(jìng)爭(zhēng)地結(jié)合E, 反應(yīng)的總方向決定于A、Q的濃度和反應(yīng)的平衡常數(shù),eg. 以NAD+或 NADP+為遞氫體的脫氫酶,eg. 肌酸激酶 (Creatine kinase),限速步驟為AEB QEP,反應(yīng)特點(diǎn): 酶同A的反應(yīng)產(chǎn)物P是在酶同第二個(gè)底物B反應(yīng)前釋放出來，作為這一過程的結(jié)果，酶E轉(zhuǎn)變?yōu)橐环N修飾酶形式F，然后再同