酶 北大生化课件 英文版.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,Chapter 2,Enzyme,Properties of enzymes,Structural features of enzymes,Mechanism of enzyme-catalyzed reactions,Kinetics of enzyme-catalyzed reactions,Inhibition of enzymes,Regulation of enzymes,Clinical applications of enzymes,Nomenclature,Contents,Section 1,Properties of Enzymes,A+B,C+D,1.1 General Concepts,spontaneous reaction only if,D,G,is negative.,at equilibrium if,D,G,is zero.,spontaneously impossible if,D,G,is positive.,Catalyzed reactions,Reactants need to pass over the energy barrier,G,+,.,Catalysts reduce the activation energy and assist the reactants to,pass over,the activation energy.,Fragile structures,of the living systems,Low kinetic energy,of the reactants,Low concentration,of the reactants,Toxicity,of catalysts,Complexity,of the biological systems,Chemical reactions in living systems are quite different from that in the industrial situations because of,Need for special catalysts,Enzymes are,catalysts,that have special characteristics to facilitate the biochemical reactions in the biological systems.,Enzyme-catalyzed reactions take place usually under,relatively mild conditions.,Enzymes,1,.2 Characteristics,Enzyme-catalyzed reactions have the following characteristics in comparison with the general catalyzed reactions:,common features:2“do”and 2“dont”,unique features:3“high”,Do not,consume themselves,:no changes in quantity and quality before and after the reactions.,Do not change the,equilibrium points,:only enhance the reaction rates.,Apply to the,thermodynamically allowable,reactions,Reduce,the activation energy,Common features,Enzyme-catalyzed reactions have very,high catalytic efficiency,.,Enzymes have a,high degree of specificity,for their substrates.,Enzymatic activities are,highly,regulated,in response to the external changes.,Unique features,Catalyst,Activation energy(cal/M),No catalyst,18,000,Normal catalyst,11,700,Hydrogen,peroxidase,2,000,1.3.a High efficiency,Accelerated reaction rates,enzyme,Non-enzymatic,rate constant,(,k,n,in s-1),enzymatic,rate constant,(,k,n,in s-1),accelerated reaction rate,Carbonic,anhydrase,10,-1,10,6,8 x 10,6,Chymotrypsin,4 x 10,-9,4 x 10,-2,10,-2,10,7,Lysozyme,3 x 10,-9,5 x 10,-1,2 x 10,8,Triose,phosphate,isomerase,4 x 10,-6,4 x 10,3,10,9,Urease,3 x 10,-10,3 x 10,4,10,14,Mandelate,racemase,3 x 10,-10,5 x 10,2,1.7 x 10,15,Alkaline,phosphatase,10,-15,10,2,10,17,Absolute specificity,Relative specificity,Stereospecificity,1.,3.b High specificity,Unlike conventional catalysts,enzymes demonstrate the ability to distinguish different substrates.There are three types of substrate specificities.,Absolute specificity,Enzymes can recognize only one type of substrate and implement their catalytic functions.,Enzymes catalyze one class of substrates or one kind of chemical bond in the same type.,Relative,specificity,Stereos,pecificity,The enzyme can act on only one form of isomers of the substrates.,Lactate,dehydrogenase,can recognize only the L-form but the D-form lactate.,Enzyme-catalyzed reactions can be regulated in response to the external stimuli,satisfying the needs of biological processes.,Regulations can be accomplished through varying the,enzyme quantity,adjusting the,enzymatic activity,or changing the,substrate concentration.,1.3,.c High regulation,Section 2,Components of Enzymes,Almost all the enzymes are,proteins,having well defined,structures,.,Some functional groups are,close enough in space,to form a portion called the,active center,.,Active centers look like a,cleft,or a,crevice,.,Active centers are hydrophobic.,2.1 Active Center,Lysozyme,Residues(colored)in the active site come from different parts of the polypeptide chain.,Binding,group:to associate with the reactants to form an enzyme-substrate complex,Catalytic,group:to catalyze the reactions and convert substrates into products,Two essential groups,The active center has two essential groups in general.,Active centers,Simple enzymes:,consists of only one peptide chain,Conjugated enzymes:,holoenzyme,=,apoenzyme,+cofactor,(protein)(non-protein),Cofactors:,metal ions;small organic molecules,2,.2 Molecular Components,Metal ions,Metal-activated enzyme:,ions necessary but,loosely bound.Often found in metal-activated enzyme.,Metalloenzymes,:Ions tightly bound.,Particularly in the active center,transfer electrons,bridge the enzyme and substrates,stabilize enzyme conformation,neutralize the anions.,Small size,and chemically stable compounds,Transferring,electrons,protons and other groups,Vitamin-like,or,vitamin-containing,molecule,Organic compounds,Loosely bind to,apoenzyme,.Be able to be separated with dialysis.,Accepting H,+,or group and leaving to transfer it to others,or vise versa.,Coenzymes,Prosthetic groups,Tightly bind through either,covalent,or many,non-covalent,interactions.,Remained,bound to the,apoenzyme,during the course of reaction.,Section 3,Mechanism of Enzyme-Catalyzed Reactions,Proximity and orientation arrangement,Multielement,catalysis,Surface effect,To understand the molecular details of the catalyzed reaction.,Lock-and-key model,Both E and S are rigid and fixed,so they must be complementary to each other perfectly in order to have a right match.,Induced-fit model,The binding induces conformational changes of both E and S,forcing them to get a perfect match.,Hexokinase,catalyzing,glycolysis,Hexokinase,the first enzyme in the,glycolysis,pathway,converted glucose to glucose-6-phosphate with consuming one ATP molecule.,Two,structural domains are connected by a hinge.,Upon binding,of a glucose molecule,domains close,shielding the active site for water.,Induced structural changes,Section 4,Kinetics of Enzyme-Catalyzed Reactions,4.1 Reaction rate,The reaction rate is defined as,the product formation per unit time.,The slope of product concentration,(P),against the time in a graphic representation is called,initial velocity,.,It is of,rectangular hyperbolic,shape.,Initial velocity,Reaction velocity curve,Intermediate state,Forming an enzyme-substrate complex,a transition state,is a key step in the catalytic reaction.,initial,intermediate,final,K,1,=rate constant for ES formation,K,2,=rate constant for ES dissociation,K,3,=rate constant for the product released from the active site,Rate constants,The mathematical expression of the,product formation,with respect to the,experimental parameters,Michaelis-Menten,equation describes the relationship between the reaction rate and substrate concentration S.,4.2,Michaelis-Menten,Equation,S E,changes of S is negligible.,K,2,is negligible compared with,K,1,.,Steady-state,:the rate of E-S complex formation is equal to the rate of its disassociation(backward E+S and forward to E+P),Assumptions,Describing a,hyperbolic,curve.,K,m,is a characteristic constant of E,S,K,m,时，,v,V,max,the substrate concentration at which enzyme-catalyzed reaction proceeds at,one-half of its maximum,velocity,K,m,is independent of E.It is determined by the,structure of E,the substrate,and,environmental conditions,(pH,T,ionic strength,),Significance of,K,m,K,m,is a characteristic constant of E.,The value of,K,m,quantifies the,affinity of the enzyme and the substrate,under the condition of,K,3,EV,E,4.3.b Effect of enzyme,4.3.c Effect of temperature,Optimal temperature,(T,o,)is the characteristic T at which an enzyme has the maximal catalytic power.,35,40,C for warm blood species.,Reaction rates increase by 2 folds for every 10,C rise.,Higher T will denature the enzyme.,4.3.d,Effect of pH,Optimal pH,is the characteristic pH at which the enzyme has the maximal catalytic power.,pH7.0 is suitable for most enzymes.,Particular examples:,pH(pepsin)=1.8,pH(,trypsin,)=7.8,Section 5,Inhibition,of,Enzyme,Inhibitors are certain molecules that can,decrease,the catalytic rate of an enzyme-catalyzed reaction.,Inhibitors can be,normal,body metabolites and,foreign,substances(drugs and toxins).,5.1 Inhibitors,The inhibition process can be either,irreversible,or,reversible,.,The inhibition can be,competitive,non-competitive,or,un-competitive,.,Inhibition processes,Inhibitors are,covalently,bound to the essential groups of enzymes.,Inhibitors cannot be removed with simple dialysis or super-filtration.,Binding can cause,a partial loss,or,complete loss,of the enzymatic activity.,5.2 Irreversible inhibition,Acetylcholine,accumulation,will cause excitement of the parasympathetic system,:omitting,sweating,muscle trembling,pupil contraction,Pesticide poisoning,Heavy metal containing chemicals bind to the,SH groups,to inactivate the enzymes.,Heavy metal poisoning,Inhibitors are bound to enzymes,non-covalently,.,The reversible inhibition is characterized by,an equilibrium,between free enzymes and inhibitor-bound enzymes.,5.3 Reversible inhibition,5.3.a Competitive inhibition,Competitive inhibitors share the,structural similarities,with that of substrates.,Competitive inhibitors,compete for the active sites,with the normal substrates.,Inhibition depends on the,affinity,of enzymes and theratio,of E to S.,Lineweaver,-Burk plot,As S increases,the effect of inhibitors is reduced,leading to,no change in,V,max,.,Due to the competition for the binding sites,K,m,rises,equivalent to the reduction of the affinity.,Inhibition features,FH,4,(tetrahydrofolate)is a coenzyme in the nucleic acid synthesis,and,FH,2,(dihydrofolate)is the precursor of FH,4,.,Bacteria cannot absorb folic acid,directly,from environment.,Bacteria use,p,-amino-benzoic acid(PABA),Glu and dihydropterin to synthesize FH,2,.,Sulfanilamide derivatives,share the structural similarity with,PABA,blocking the FH,2,formation as a competitive inhibitor.,Example-1:competitive inhibitor,Example-2:competitive inhibitor,5.3.b Non-competitive inhibition,Inhibitors bind to,other sites,rather than the active sites on the free enzymes or the E-S complexes.,The,E-I,complex formation,does not,affect the binding of substrates.,The E-I-S complexes,do not proceed,to form products.,Reducing the E-S,V,max,;unchanged,K,m,.,5.3.c Uncompetitive inhibition,Uncompetitive inhibitors bind only to the,enzyme-substrate,complexes.,The E-I-S complexes do not proceed to form products.,The E-I-S complexes do not backward to the substrates and enzymes.,This inhibition has the effects on reducing both,V,max,and,K,m,.,Commonly in the,multiple substrate,reactions.,type,binding target,K,m,V,max,Competitive,E only,=,Noncompetitive,E or ES,=,Uncompetitive,ES only,Summary of inhibition,Activators are the compounds which bind to an enzyme or an enzyme-substrate complex to,enhance the enzymatic activity,without being modified by the enzymes.,Activator,Metal ions,essential activators,:no enzymatic activity without it,Mg,2+,of hexokinase,non-essential activators,:enhancing the catalytic power.,Activators,Enzymatic activity is a measure of the capability of an enzyme of catalyzing a chemical reaction.,It directly affects the reaction rate.,International unit(IU),:the amount of enzyme required to convert 1,mol of substrate to product per minute under a designated condition.,Enzymatic activity,Determination of the enzymatic activity requires proper treatment of enzymes,excess amount of substrate,optimal T and pH,One,katal,is the amount of enzyme that converts 1 mol of substrate per second.,IU=16.67,10,-9,kat,In addition to enzymes,other chemical species often participate in the catalysis.,Cofactor,:chemical species required by inactive,apoenzymes,(protein only)to convert themselves to active,holoenzymes,.,2,.2 Molecular Components,Cofactors,Activator ions:,loosely and reversibly bound,often participate in the binding of substrates.,Metal ions of,metalloenzymes,:tightly bound,and frequently participate directly in catalytic reactions.,Essential ions,Transfer electron,Linkage of S and E；,Keep conformation of E-S complex,Neutralize anion,Function of metal ions,Act as group-transfer reagents to supply active sites with reactive groups not present on the side chains of amino acids,Cosubstrates,:,Prosthetic groups,:,CoenzymesThe,substrates,in nature.,Their,structures,are altered for,subsequent reactions,.,Shuttle,mobile metabolic groups among different enzyme-catalyzed reactions.,Cosubstrates,Supply the,active sites,with reactive groups not present on the side chains of AA residues.,Can be either,covalently,attached to its,apoenzymes,or through many,non-covalent,interactions.,Remained,bound to the enzyme,during the course of the reaction.,Prosthetic groups,Metabolite coenzymes,:they are synthesized from the common metabolites.,several NTP,ATP(most abundant),UDP-glucose,Vitamin-derived coenzymes,:they are derivatives of vitamins,and can only be obtained from nutrients.,NAD and NADP,+,FAD and FMN,lipid vitamins,Coenzymes,Until recently,all the enzymes are known to be proteins.,Ribonucleic acids also demonstrate the catalytic ability.,Ribozymes,have the ability to self-cleave.,They are highly conservative,an indication of the biological evolution and the primary enzyme.,2,.3,Ribozyme,Family of serine protease,5.3.a Competitive inhibition,5.3.b Non-competitive inhibition,5.3.c Uncompetitive inhibition,Section 6,Regulation of Enzyme,Many biological processes take place at a specific,time;,at a specific,location,and at a specific,speed,.,The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency.,The key step of this process is to regulate either the enzymatic,activity,or the enzyme,quantity,.,Maintenance of an ordered state,in a timely fashion and without wasting resources,Conservation of energy,to consume just enough nutrients,Rapid adjustment,in response to environmental changes,Reasons for regulation,Controlling an enzyme that catalyzes the,rate-limiting reaction,will regulate the,entire metabolic pathway,making the,biosystem,control more efficient.,Rate limiting reaction is the reaction whose rate set by an enzyme will dictate the whole pathway,namely,the slowest one or the“bottleneck”step.,Zymogen,activation,Allosteric,regulation,Covalent modification,6,.1 Regulation of E Activity,Certain proteins are synthesized and secreted as an,inactive,precursor of an enzyme,called,zymogen,.,Selective,proteolysis,of these precursors leads to,conformational changes,and,activates,these enzymes.,It is the conformational changes that either,form an,active site,of the enzyme or,expose the active site,to the substrates.,6,.1.a,Zymogen,activation,Hormones,:,proinsulin,Digestive proteins,:,trypsinogen,Funtional,proteins,:factors of blood clotting and clot dissolution,Connective tissue proteins,:,procollagen,Wide varieties,Activation of,chymotrypsin,A cascade reaction in general,To protect the zymogens from being digested,To exert function in appropriate time and location,Store and transport enzymes,Features of,zymogen,activation,Allosteric,enzymes,are those whose activity can be adjusted by,reversible,non-covalent,binding of a specific modulator to the regulatory sites,specific sites on the surface of enzymes.,Allosteric,enzymes are norma