生物化学教学课件：§5 Enzyme

liguofu5.1 Introduction5.2 How Do Enzymes Play Specificity 5.3 How Do Enzymes Accelerate Reaction5.4 Enzyme examples5.5 Enzyme Kinetics5.6 Enzyme Regulation5 Enzyme1.Brief History2.Classes&Name3.Composition4.General FeaturesliguofuIn 1810,Joseph Gay-Lussac(约瑟夫约瑟夫盖盖吕萨吕萨克，克，1778.12-1850.5，法，法国化学家和物理学家国化学家和物理学家)found that ethanol and CO2 were the principal products of sugar decomposition by yeast.Brief HistoryliguofuBrief History(cntd)In 1835,Jacob Berzelius(雅各布雅各布贝采利乌斯贝采利乌斯,1779.8-1848.8，瑞典化，瑞典化学家学家)pointed out that an extract of malt(known as diastase)catalyzes the hydrolysis of starch more efficiently than does sulfuric acid.liguofuBrief History(cntd)In the mid-19th century,Louis Pasteur(1822-1895,法国微生物学法国微生物学家家)proposed that the processes of fermentation could only occur in living cells(vital force);Others,however,notably Justus von Liebig(尤斯蒂斯尤斯蒂斯冯冯李比希李比希,1803-1873,德德国化学家国化学家),argued that biological processes are caused by the action of chemical substances that were then known as“ferments.”liguofuBrief History(cntd)In 1878,Wilhelm Friedrich Khne(威廉威廉弗里德里希弗里德里希屈屈内内,1837.31900.6，德国生，德国生理学家理学家)coined the word “enzyme”(Greek:en,in+zyme,yeast)to emphasize that there is something in yeast,as opposed to the yeast itself,that catalyzes the reactions of fermentation.liuofu1941现代生物现代生物化学化学18911940酶学时代酶学时代18401890生理化学生理化学18001839有机化学有机化学17721799化学革命化学革命生物化学生物化学酶学酶学哲学哲学生理学生理学化学化学生理化学生理化学有机化学有机化学物理学物理学现代现代生物化学生物化学生物化学发展的脉络生物化学发展的脉络Brief History(cntd)liguofuBrief History(cntd)Eduard Buchner(毕希毕希纳纳,1860.5-1917.8):德国德国化学家化学家,1894年，与其兄年，与其兄弟一起发现酵母细胞的弟一起发现酵母细胞的萃取液即可致发酵作用。萃取液即可致发酵作用。1897年从这些萃取液分年从这些萃取液分离出有效的成份，称为离出有效的成份，称为zymase，从而导致对酵，从而导致对酵素的了解，并因此曾获素的了解，并因此曾获1907年诺贝尔化学奖年诺贝尔化学奖liguofuBrief History(cntd)In 1894,Emil Fischers(费歇费歇尔尔,1852.10-1919.7，德国化学，德国化学家家,1902年诺贝尔化学奖年诺贝尔化学奖)discovery that glycolytic enzymes can distinguish between stereoisomeric sugars led to the formulation of his lock-and-key hypothesis.liguofuBrief History(cntd)In 1926,James Sumner(詹姆斯詹姆斯萨姆纳萨姆纳,887.11-1955.8,美国化学家美国化学家,1946年诺贝尔化学奖年诺贝尔化学奖)crystallized the first enzyme,jack bean（洋（洋刀豆）刀豆）urease,and these crystals consist of protein.liguofuBrief History(cntd)Since Sumners preparations were somewhat impure,however,the protein nature of enzymes was not generally accepted until the mid-1930s,when John Northrop(1891.7-1987.5 美国生化学家美国生化学家)showed that there is a direct correlation between the enzymatic activities of crystalline pepsin,trypsin,and chymotrypsin and the amounts of protein present.liguofuBrief History(cntd)1963年，年，牛胰核糖核酸酶牛胰核糖核酸酶成为第一个被完全确定成为第一个被完全确定一级结构的酶一级结构的酶:Smyth DG,Stein WH,Moore S.The sequence of amino acid residues in bovine pancreatic ribonuclease:revisions and confirmations.J Biol Chem.1963,238:227-2341965年年,卵清蛋白溶菌酶卵清蛋白溶菌酶成为第一解析三维空间结成为第一解析三维空间结构的酶：构的酶：Blake CC,Koenig DF,Mair GA et al.Structure of Hen Egg-White Lysozyme:A Three-dimensional Fourier Synthesis at 2 Resolution,Nature,1965,206:757-761liguofuClasses&NameNo.ClassType of reaction catalyzed1Oxidoreductases(氧化还原酶氧化还原酶)Transfer of electrons(hydride ions or H atoms)2Transferases(转移酶转移酶)Group transfer reactions3Hydrolases(水解酶水解酶)Hydrolysis reactions(transfer of functional groups to water)4Lyases(裂合酶裂合酶)Addition of groups to double bonds,or formation of double bonds by removal of groups5Isomerases(异构酶异构酶)Transfer of groups within molecules to yield isomeric forms6Ligases(连接酶连接酶)Formation of C-C,C-S,C-O,and C-N bonds by condensation reactions coupled to ATP cleavageliguofuTrivial(common)name：adding the suffix“-ase”to the name of their substrate or to a word/phrase describing their activityUrease(hydrolysis of urea).Transaminase(transfer amino group).RNA polymerase But many enzymes are named before this rule was established(e.g.,pepsin,trypsin).Classes&Name(cntd)liguofuHNADHPyruvateNADLactateLactate dehydrogenase(lactate:NAD+oxidoreductase)E.C.1.1.1.27Class:OxidoreductaseEnzyme CommissionSub-Class:Acting on the primary&secondary alcoholsSub-Sub-Class:NAD+as electron acceptorSpecific enzymewithin sub-sub-classSystemic nameliguofu辅因子依赖性酶辅因子依赖性酶(Cofactor-dependent)全酶全酶(holo)=脱辅基酶蛋白脱辅基酶蛋白(apo)+辅因子辅因子(cofactor)非辅因子依赖性酶非辅因子依赖性酶(Cofactor-independent)酶酶辅因子辅因子(cofactor)无机无机(inorganic):如金属离子如金属离子有机有机(organnic)辅基辅基(prosthetic group)：与酶结合紧密（共价或与酶结合紧密（共价或非共价非共价)辅酶辅酶(coenzyme)：与酶：与酶可逆结合，常为维生素可逆结合，常为维生素的衍生物的衍生物CompositionliguofuliguofuliguofuGeneral FeaturesEnzyme(biocatalyst)contrast with chemical catalyst High reaction ratesMild reaction conditionsGreat reaction specificityCapacity for controlliguofuliguofu5.1 Introduction6.2 How Do Enzymes Play Specificity 5.3 How Do Enzymes Accelerate Reaction5.4 Enzyme examples5.5 Enzyme Kinetics5.6 Enzyme Regulation5 Enzyme1.Specificity in shape2.Specificity in chiralityliguofuSpecificity between proteins and ligandsliguofuSpecificity in shapeThe active site of an enzyme is compatible in both shape and interaction force with its substrate.A few enzymes:only one substrate.Most enzymes:a small range of related substrates.Some enzymes,particularly digestive enzymes:a large range of related substrates.Hydrophobic groupsliguofuSpecificity in chiralitylAn enzyme catalytic reaction is a chiral reaction because the active site is a chiral enviromentlIt is easy to understand a chiral reaction if the substrate is a chiral moleculelHowever，there are a lot of achiral molecules as enzyme substrates in cell,and the products are chiral molecules.Why?Prochirality!liguofuWhat is prochirality?If a molecule can be converted from achiral to chiral in a single step,the molecule is prochiral or has prochirality.R/S systemliguofuProchiral differentiation of enzymeThe specific attachmentof a prochiral center to an enzyme binding site permits the enzyme to differentiate between prochiral groupsEthanols two methylene H atoms may be distinguished if the molecule is held in some sort of asymmetric jig.The substrate-binding sites of enzymes are,of course,just such jigs because they immobilize the reacting groups of the substrate on the enzyme surface.liguofu5.1 Introduction5.2 How Do Enzymes Play Specificity 5.3 How Do Enzymes Accelerate Reaction5.4 Enzyme examples5.5 Enzyme Kinetics5.6 Enzyme Regulation5 Enzyme1.A macroscopic view Enzymes decrease the activation energy2.A microscopic view How do enzymes decrease the activation energy?liguofu A macroscopic viewThermodynamics defines the reaction rates&equilibrialneqoKRTGSPKeqFrom thermodynamicsSkV RTGehTk*Boltzmann constant Plancks constant Rate constantliguofuA macroscopic viewEnzymes decrease the activation energylneqoKRTGRTGehTk*oGliguofuES complexE-transitionstate complexnon-covalent interactions between enzyme and substrate are optimized in the transition state Transition theory liguofu(1)Binding energy(2)Specific Catalytic GroupsGeneral Acid-Base CatalysisCovalent CatalysisMetal ion catalysisA microscopic view How do enzymes decrease the activation energy?liguofu Induced conformational changeunder this condition,the following actions occur.Desolvation Entropy reduction Electorn redistributionBinding energy can be used to overcome these barriersProximity orientationliguofuInduced fit in hexokinaseAn example for the transition theory of enzymes liguofuDesolvationliguofuRate enhancement by entropy reductionliguofuWhen proton transfer to or from H2O is faster than the rate of breakdown of intermediates,the presence of other proton donors or acceptors does not increase the rate of the reaction.Specific Catalytic Groups/General Acid-Base Catalysis+OH-H2OH+HOH-H2OB:AHBH+A-When proton transfer to or from H2O is slower than the rate of breakdown of intermediates,only a fraction of the intermediates formed are stabilized.The presence of alternative proton donors(HA)or acceptors(B:)increases the rate of the reaction.liguofuAmino acids in general acid-base catalysisliguofuSpecific Catalytic GroupsliguofuCovalent and acid-base catalysis work together liguofuSpecific Catalytic Groups/metal ion catalysis liguofu5.1 Introduction5.2 How Do Enzymes Play Specificity 5.3 How Do Enzymes Accelerate Reaction5.4 Enzyme examples5.5 Enzyme Kinetics5.6 Enzyme Regulation5 Enzyme1.Chymotrypsin2.Serine protease family3.Other protein hydrolasesHis57Asp102Ser195Crystal structure(1967)revealed a catalytic triad:Ser195,His57,Asp102ChymotrypsinliguofuHNphenylNOliguofuAsp102 functions only to orient His57.liguofuliguofuliguofuliguofuliguofuliguofuliguofuAnimation of Chymotrypsin catalytic Mechanism liguofupH dependence of chymotrypsin-catalyzed reactionsWhy?Effect on the acid/base catalysis of His57Effect on the conformation of Phenyl pocketliguofuSerine Protease is a large family of enzymes whose catalytic mechanism is based on an active-site serine residue，including：chymotrypsin trypsin elastase thrombin（凝血酶）（凝血酶）subtilisin（枯草杆菌蛋白酶）（枯草杆菌蛋白酶）plasmin（血纤维蛋白溶酶）（血纤维蛋白溶酶）Serine protease familyA catalytic triad has been found in all serine proteases:the Ser is thus converted into a potent nucleophile(subtilisin has no homology with other Ser protease members,but has the triad)liguofuliguofuliguofuliguofuliguofuliguofuThe specificity of serine proteases is determined by the structural features of a substrate binding pocketliguofuAspartyl Protease:Renin（血管紧张肽原酶）（血管紧张肽原酶）AspOther protein hydrolases(1)liguofuOther protein hydrolases(2)liguofuMetalloproteaseThermolysin（嗜热菌蛋白酶）（嗜热菌蛋白酶）Other protein hydrolases(3)liguofu5.1 Introduction5.2 How Do Enzymes Play Specificity 5.3 How Do Enzymes Accelerate Reaction5.4 Enzyme examples5.5 Enzyme Kinetics5.6 Enzyme Regulation5 Enzyme1.Steady state&Pre-steady state 2.One substrate kinetics3.Two substrates kineticsliguofuSteady state kineticsPre-steady state kineticsliguofuOne substrate kinetics:Michaelis-Menten equation 合合理理简简化化Kcat代表代表“暗箱暗箱”的表的表观速率常数，是各正向观速率常数，是各正向速率常数的函数速率常数的函数.如果如果“暗箱暗箱”中只有一个限中只有一个限速步骤，则速步骤，则kcat 近似于近似于该步的正向速率常数。该步的正向速率常数。ES代表代表“暗箱暗箱”中中从从ES1到到EPm各种复各种复合物；合物；稳态的开始阶段稳态的开始阶段,P很低很低,形成复合物的形成复合物的逆反应可忽略逆反应可忽略liguofu)ES(ES)S(E11catkkk稳态时，mcatKkkkSES)/(SESES11liguofumKSSEkESkVcatcat0在底物过量的情况下，酶被底物所饱和，在底物过量的情况下，酶被底物所饱和，ES=EmaxEkVcatmKSSVVmax0This is the Michaelis-Menten EquationliguofuWhen S KmKm is equivalent to the substrate concentration at which V0 is Vmax/2mKSSVVmax0Most enzymes(except the regulatory enzymes)have been found to follow the Michaelis-Menten kineticsliguofu对对kcat、Km、kcat/Km的说明的说明表观速率常数表观速率常数Kcat：又称酶的转换数又称酶的转换数(turn number)，反映了一个酶的催化效率反映了一个酶的催化效率.kcat/Km：反映酶与底物受扩散速率限制的匹配程度，反映酶与底物受扩散速率限制的匹配程度，绝大多数高效酶的绝大多数高效酶的kcat/Km比值为比值为108-109 M-1S-1.liguofuliguofu支持支持 transition state theory 的证据的证据对底物结构的变化主要影响kcat而对Km影响较小，如糜蛋白酶.Transition-state analogs bind to enzymes 102-106 times more tightly than normal substrates.The idea of catalytic antibodies was also suggested by this theory(Jencks,1969)and approved to be correct(Lerner and Schultz,1980s).liguofuChymotrypsin 三种底物的三种底物的kcat,Km 比较比较liguofu磷酸（盐）可作为酯或碳酸酯化物水解的磷酸（盐）可作为酯或碳酸酯化物水解的过渡态类似物，通过免疫获得催化性抗体过渡态类似物，通过免疫获得催化性抗体liguofuDouble reciprocal plot(i.e.,the Lineweaver-Burk Plot).如何求如何求Km和和kcatliguofuEach substrate will have one characteristic Km value.Ternary complex may or may not be formed for the bisubstrate reactions depending on the mechanism.Steady-state kinetics can often help distinguish these two mechanisms.Two substrates kineticsliguofuliguofuKeeping【S2】constant,the double reciprocal plots made by varying【S1】Goto 96 liguofuPing-Pong(or double displacement)mechanismliguofuKeeping【S2】constant,the double reciprocal plots made by varying【S1】Goto 94 liguofu5.1 Introduction5.2 How Do Enzymes Play Specificity 5.3 How Do Enzymes Accelerate Reaction5.4 Enzyme examples5.5 Enzyme Kinetics5.6 Enzyme Regulation5 Enzyme1.Artificial inhibition2.Physiological regulationliguofuArtificial inhibitionSuch inhibitors are important pharmaceutical agents and useful in understanding the action mechanism of enzymesInhibitionIrreversible Reversible CompetitiveUncompetitiveMixed Group-specificAffinitySuicideIrreversible inhibitors(inactivator)：chemically modify or form tight noncovalent interactions with functional groups in the active site of enzymes.liguofu Group-specific inactivatorSH特异性灭活剂特异性灭活剂OH特异性灭活剂对糜蛋白酶的灭活特异性灭活剂对糜蛋白酶的灭活25个个Ser之一之一liguofuTPCK alkylates His57 of chymotrypsin(It does not occur when chymotrypsin is denatured in urea)Affinity inactivatorsliguofu青霉素（青霉素（penicillin）类的抗菌机理）类的抗菌机理e.g.1liguofu革兰氏阳性和阴性菌细胞壁中的肽聚糖（革兰氏阳性和阴性菌细胞壁中的肽聚糖（peptidoglycan）liguofu肽聚糖的结构肽聚糖的结构L-ala D-gluL-lys D-alaD-alaGly-Gly-Gly-Gly-GlyPeptidoglycan in S aureus(葡萄球菌)cell wall liguofu转肽酶催化肽聚糖合成中的转肽交联反应转肽酶催化肽聚糖合成中的转肽交联反应liguofu青霉素与转肽酶底物结构非常相似，进入青霉素与转肽酶底物结构非常相似，进入活性中心，共价修饰活性中心，共价修饰Ser，致转肽酶失活，致转肽酶失活liguofu细菌进化产生抗药性：细菌进化产生抗药性：-lactamase-lactamase：内酰胺酶内酰胺酶水解内酰胺键，导致青霉水解内酰胺键，导致青霉素类抗菌素失灵素类抗菌素失灵liguofue.g.2抗抗HIV蛋白水解酶药物蛋白水解酶药物HIV生生活活史史及及药药物物的的靶靶点点liguofuHIV蛋白水解酶的机理蛋白水解酶的机理HIV protease属于Asp蛋白水解酶家族，主要识别水解Phe的羧基和Pro的氨基形成的肽键liguofu下列化合物都是下列化合物都是HIV protease底物的过渡态类似物，底物的过渡态类似物，虽然不能共价修饰该酶的活性中心，但与活性中心虽然不能共价修饰该酶的活性中心，但与活性中心结合非常紧密，也属于不可逆亲和抑制剂结合非常紧密，也属于不可逆亲和抑制剂liguofuSuicide inactivator：不仅象酶的天然底物，并且可被酶催化转化，但转化的中间产物可修酶的活性基团，固曰“酶的”自杀灭活剂，如开发的针对具有青霉素抗药性细菌的药物clavulanic acid（克拉维酸）。Clavulanic acidliguofuClavulanic acid（克拉维酸）灭活-lactamase的机理的机理liguofu1max0EIIEKKISKSVVfreeIIm liguofuCompetitive inhibitors alters the Km but not the Vmax of enzymesliguofuInhibitor only binds to the ES complex,1max0ESIIESKKISKSVVIIm liguofuUncompetitive inhibitors alter both the Km and the Vmax of an enzymeGoto 78 liguofumax0SKSVVmMixed inhibition liguofu Mixed inhibitors alter both the Km and the Vmax of an enzymeGoto 76liguofuNoncompetitive inhibitionis a special mixed inhibition when =Noncompetitive inhibitors alters the Vmax but not the Km of enzymes)(max0SKSVVmliguofuPhysiological regulation1.Allosteric regulation(noncovalent modifications,reversible);2.Covalent modifications(reversible);3.Proteolytic cleavage(irreversible).4.(Gene regulation:changing the amount of specific enzymes).liguofu1.Allosteric regulationPositive heterotropic effectNegative heterotropic effectNegative feedbackliguofuAllosteric enzymes can has both positive and negative modulators,e.g.ATCase(Aspartate transcarbamoylase,天冬氨酸转氨甲酰酶),Why?liguofuAnswerliguofu如：如：糖原磷酸糖原磷酸化酶的活性调化酶的活性调节：通过节：通过磷酸磷酸化化和和去磷酸化去磷酸化调节活性调节活性2.Covalent modificationsliguofu多位点磷酸化多位点磷酸化修饰对酶活性修饰对酶活性的调节更加精的调节更加精细细，如糖原合如糖原合成酶有成酶有9 9个磷酸个磷酸化位点，有化位点，有9 9中中蛋白激酶参与蛋白激酶参与其活性调节其活性调节。liguofuX-any residue;B-any hydrophobic residue;S,T,Y-磷酸化残基 Ser,Thr,Tyr.常见蛋白激酶的识别位点和修饰的氨基酸残基常见蛋白激酶的识别位点和修饰的氨基酸残基liguofu其他常见修饰基团及其修饰的氨基酸残基其他常见修饰基团及其修饰的氨基酸残基主要是一些酶原的激活主要是一些酶原的激活.3.Proteolytic cleavageliguofu体内多种机制直接或间接调节一个体内多种机制直接或间接调节一个酶的活性，如糖原磷酸化酶的活性酶的活性，如糖原磷酸化酶的活性调节调节liguofuSome other concepts IsoenzymeRibozyme Artificial enzymeImmobilized enzymeSpecific activityliguofuThe EndThe End