1、Chapter 3 Enzymes Enzymes are biocatalysts with high specificity and highly catalytic efficency produced by living cells.Besides proteins,ribozymes and deoxyribozymes are also enzymes.What is enzyme?（1）producing site:living cells（2）property:mostproteins a fewRNA DNA（3）function:biochemical catalyzati
2、on3.1 Structure and Function of Enzymes 3.2 Properties and Catalytic Mechanisms of Enzymes3.3 Kinetics of Enzyme-Catalyzed Reactions3.4 Regulation of Enzyme Activity3.5 Nomenclature and Classification of Enzymes3.6 Clinical Applications of Enzymes OUTLINE3.1 Structure and Function of Enzymes3.1.1 st
3、ructure of enzymesenzymesSimple enzymesproteinsapoenzymeprosthetic groupcoenzymeConjugated enzymes(holoenzyme)cofactor3.1.1.1 composition of Enzyme MoleculesHoloenzyme=Apoenzyme+Cofactors uProsthetic group:tightly bond with apoenzyme and can not be removed by dialysis（分离，透析）（分离，透析）or ultrafiltration
4、.FAD,metal,etc.uCoenzyme:loosely bond with apoenzyme and can be removed by dialysis or ultrafiltration.NAD,NADP,etc.Cofactors are small organic（有机的）（有机的）molecules or inorganic（无机的）（无机的）ions required for enzyme activities.coenzymesmall organic moleculesmetal ionIron,magnesium,cobalt,manganeseFunction
5、s of small organic molecules：act as carriers to transfer electrons,protons（质子）（质子）or other groups.CoenzymesTransferred groupsVitamins Nicotinamide adenine dinucleotide(NAD+)H+,eV-ppNicotinamide adenine dinucleotide phosphate(NADP+)H+,eV-ppFlavin adenine dinucleotide(FAD)HV-B2Flavin mononucleotide(FM
6、N)HV-B2Thiamine pyrophosphate(TPP)-CHO(aldehyde)V-B1Coenzyme A(CoA)R-CO-(acyl)Pantothenic acidPyridoxal phosphate-NH2V-B6BiotinCO2BiotinTetrahydrofolate“C”(one carbon unit)Folic acid5-deoxyadenosyl cobalamineCH3、-CnH2n+1(alkyl)V-B12TABLE 3-1 Cofactors of enzymescoenzymesmall organic moleculesmetal i
7、onIron,magnesium,cobalt,manganese(Fe)(Mg)(Co)(Mn)metalloenzyme bind metal ion tightly metal ion is retained throughout purification metal-activated enzyme bind metal ion less tightly metal ion is essential for activityFunctions of metal ion：stabilize enzyme conformation participate in reaction by tr
8、ansferring electrons approximation and orientation of reactants as bridge between substrate and enzyme neutralize negative charges to decrease electrostatic repulsion monomeric enzyme：only contain a polypeptide chain with tertiary structure.oligomeric enzyme：contain two or more polypeptide chains as
9、sociated by noncovalent bonds.3.1.1.2 monomeric enzymes,oligomeric enzymes and multienzyme complexmultienzyme complex：different enzymes catalyze sequential reactions in the same pathway are bound position of pyruvate dehydrogenase complexEnzymes cofactorsPyruvate dehydrogenase TPP Dihydrolipoyl tran
10、sacetylase lipoic acid&.CoADihydrolipoyl dehydrogenase FAD&.NAD+Pyruvate+CoA+NAD+acetyl CoA+CO2+NADH+H+Pyruvate dehydrogenase complexmultifunctional enzyme（tandem enzyme）：）：a single polypeptide chain with multiple activities.Fatty acid synthesis multifunctional enzyme of mammal This enzyme consists
11、of seven enzyme activities in a single polypeptide chain encoded by a gene.Fatty acid synthase3.1.2.Active site of an enzymeEssential groups：Some chemical groups essential for maintaining the enzyme activity are termed essential groups.Primary structure and spatial structure of chymotrypsinActive si
12、te:The active site is the region of the enzyme that contains some chemical groups for binding substrates and transforming it into products.The active site is a three-dimensional entity,often a cleft or crevice on the surface of the protein,in which the substrate is bound by multiple weak interaction
13、s.Three-dimensional structure Substrate bindingsubstrate Essential groups outside of active site Binding groups catalytic group active siteActive site of enzymes Essential groupsactive siteoutside of active site：maintain conformation of enzymeSubstrate-binding groupscatalytic groupdetermine specific
14、ity for substratedetermine characteristics of catalyzation 3.1.3 Isoenzymes Isoenzymes:Multiple forms of an enzyme which catalyze the same reaction,but differ from each other in their amino acid sequence,physicochemical properties and immuno-characters.Lactate dehydrogenase(LDH)H subunitM subunitTet
15、ramer LDH (M.W.130,000)Subunit:H (Chr12)M (Chr11)pyruvateLactateLDHorigin LDH5 LDH4 LDH3 LDH2 LDH1Cellulose Acetate Membrane Electrophoresis result of LDH isoenzymes_+anodecathodeH4M4H1M3H2M2 H3M1Tissue distribution specificityLDH1(H4)LDH2(H3M)LDH3(H2M2)LDH4(HM3)LDH5(M4)Physiological significance of
16、 isoenzymes regulation of metabolism(different tissue or different developmental stage).diagnosis of different diseases.Enzyme activityinfarctionNormalLiver DiseaseLDH isoenzymogram in serum 3.2 Properties and Catalytic Mechanisms of EnzymesThe essential properties of both enzymes and inorganic cata
17、lysts:1.Only catalyze thermodynamic reactions.2.Remain unchanged of their quality and quantity.3.Increase the rate of reaction,but can not alter the equilibrium constant of the reaction.4.Decrease the activation energy of reaction to increase the velocity of the reaction 3.2.1 Properties of enzyme c
18、atalyzed reactions3.2.1.1 Highly Catalytic Activity of Enzymes a)In general,enzyme-catalyzed reactions are 1081020 times faster than the corresponding uncatalyzed reactions，1071013 times faster than inorganic catalytic reactions.b)The temperature needed is not high.c)Enzymes decrease the activation
19、energy more effectively than inorganic catalysts.properties of enzymes that are different from inorganic catalysts:example:2H2O2 2H2O+O2 catalysts efficiency Fe2+6 10-4mol/g.S heme 6 10-1 mol/mol.S peroxydase 6 106 mol/mol.SSpecificity：Specificity refers to the ability of an enzyme to discriminate b
20、etween two competing substrate and catalyze one specific reaction.3.2.1.2 Highly Specificity of Enzymes Absolute specificity:An enzyme just acts on a special substrate.For example,urease only hydrolyzes urea to form NH3 and CO2.Relative specificity:An enzyme acts on a group of related substrates or
21、one types of chemical bond.For example,peptidase and hexokinase,etc.Stereospecificity:An enzyme acts on a single stereoisomer.For example,LDH hydrolyzes L-lactate.a.Compartmentationb.Isoenzymesc.Allosteric regulation(3.4)d.Covalent modification(3.4)e.Induction and repression(3.4)3.2.1.3 Activities o
22、f enzymes can be regulated3.2.2.Catalytic Mechanisms of enzymesEnzymes accelerate reactions by decreasing the activation energy.For a biochemical reaction to proceed,the energy barrier needed to transform the substrate molecules into the transition state has to overcome.The transition state has the
23、highest free energy in the energy pathway.3.2.2.1 Formation of Enzyme-substrate Complex and Induced-fit Hypothesis The combination of substrate and enzyme forces the substrate to become the transition state.activation energy：the free energy required to promote reactants from the ground state to the
24、transition state in chemical reactions.Energy diagram for catalyzed and uncatalyzed reactions Enzyme-substrate complex How does an enzyme lower the activation energy of a reaction?Formation of the enzyme-substrate complex(ES)E+SESE+PTransition statemodel-enzyme bind its substratepInduced fit model h
25、ypothesis Daniel E.Koshland,1958How does the ES form?Induced fit modelInduced fit modelActive sites in the uninduced enzymeBinding of the first substrate(gold)induces a conformational shift that facilitates binding of the second substrate(blue),with far lower energy than otherwise required.When cata
26、lysis is complete,the product is released,and the enzyme returns to its uninduced state.Hexokinase-D-glucose complexHexokinaseInduced fitInduced-fit model of carboxypeptidase substrate(1)Proximity and orientation effects 3.2.2.2 Catalytic mechanisms of enzymesFIGURE 3-7 Proximity effects and orienta
27、tion arrange(2)Electrostatic effects The active sites of enzymes are often hydrophobic.Water is largedly excluded from the active site because of the low dielectric constant.This increases the electrostatic interactions between enzymes and substrates and accelerate velocity of the reaction.(3)Acid-b
28、ase catalysis Side chain groups in enzyme active sites,act as proton donors or acceptors are termed general acids or general bases.The transition state is stabilized and the rate of reaction is increased by adding or removing proton from reactants.These chemical groups are imidazole group,amino grou
29、p,carboxyl group,and so on.(4)Covalent catalysis The attack of nucleophilic or electrophilic group in the enzyme active site upon the substrate results in covalent binding of substrate to the enzyme as an intermediate in the cause of catalysis.Ser-OHCH2S：HCH2O：HCys-SHCH2C=CHHNNCH：His-imidazolenucleo
30、philic3.3 Kinetics of Enzyme-catalyzed ReactionsKinetics of Enzyme-Catalyzed Reactions:is a study on the rate of enzyme-catalyzed reactions and the factors affected the reaction rate.The factors affected the rate of enzyme-catalyzed reactions：substrateenzymetemperaturepHactivatorinhibitor The veloci
31、ty of a biochemical reaction is defined as the change in the concentration of a substrate or product per unit time.The initial velocity（初速率,V0)is the reactive velocity during the first few seconds of the reaction,and is determined as the slope of the curve through the linear phase in the plot of pro
32、duct concentrations versus reactive time Enzyme activity may be expressed in a number of ways,the commonest is by theV0.There are 2 standard units of enzyme activity:International unit(IU)and the katal(kat)1 IU：the amount of enzyme that produce 1 mol of product per min at 25oC under optimal conditio
33、ns.1kat:the amount of enzyme that converts 1 mol of substrate to product per second at 25oC under optimal conditions.1 IU=16.67x10-9 kat Specific activity is the number of units per milligram of protein(units/mg).The specific activity is a measure of the purity of an enzyme.3.3.1.The effect of subst
34、rate on the rate of enzyme-catalyzed reactionsPreconditions:Initial velocity(V0)S is much greater than ERectangular hyperbola plotFirst order reaction:The rate of the reaction is directly proportional to S only when S is low.Zero-order reaction:When S is sufficiently high,the velocity approach maxim
35、um velocity(Vmax).Mixed-order reaction S+EESE+Pk1k2k3K1=rate constant for ES formationK2=rate constant for ES dissociationK3=rate constant for product formation and release from the active site Enzyme-catalyzed reaction processKm：Michaelis constantMichaelis-Menten Equation(1913)v=Vmax SKm+S K2+K3 Km
36、=K1 SKSVVmm+=V=VmKmSFirst order Mixed-orderZero-orderV Vm当当 S Km时时,当当 S E时时,V=k3 EVE03.3.3.The effect of temperature on the rate of enzyme-catalyzed reactionsEnzyme activityTemperature(C)optimum temperatureOptimum temperature:Temperature at which it operates at maximal efficiency.It is not a charact
37、eristic constant of an enzyme.Bell-shaped curve3.3.4.The effect of pH on the rate of enzyme-catalyzed reactionsOptimum pH:The pH value at which an enzymes activity is maximal.It is not a characteristic constant of an enzyme.PepsinAmylaseEnzyme activitypHAcetylcholinesterase3.3.5 The effect of inhibi
38、tors on the rate of enzyme-catalyzed reactionsInhibitor:any molecule which acts directly on an enzyme to lower its catalytic rate without denaturation.irreversible inhibitionreversible inhibitioncompetitive inhibitionnon-competitive inhibitionuncompetitive inhibition Classification of inhibitions3.3
39、.5.1 Irreversible enzyme inhibition Irreversible inhibitors usually bond covalently to the enzyme,often to a side chain group in the active site.Inhibition can not be reversed by dialysis or ultrafiltration.Example 1 organophosphorous compoundsRegeneration of active enzyme pyridine aldoxime methylio
40、dide(PAM)Example 2 compounds with heavy metal ions C Cl l H HS S S SC Cl l-C CH HC CH H-A As s E E C Cl l-C CH H=C CH H-A As s E E2 2H HC Cl l C Cl l H HS S S S 路路易易士士气气 巯巯基基酶酶 失失活活的的酶酶 S S C CH H2 2-S SH H H HS S C CH H2 2-S SC Cl l-C CH H=C CH H-A As s E E C CH H-S SH H E E C CH H-S S A As s-C CH
41、HC CH H-C Cl l S S C CH H2 2-O OH H H HS S C CH H2 2O OH H 失失活活的的酶酶 巯巯基基酶酶 和和砷砷剂剂结结合合物物(Hg2+、Ag+、As3+ect.)-SH enzymelewisiteInhibited enzymeRegeneration of active enzyme2,3-dimercaptopropanol(BAL)BALInhibited enzymeCompond with Asactive enzymeExample 3 Antibiotic penicillin3.3.5.2 reversible enzyme
42、inhibition Reversible inhibitor can dissociate from the enzyme because it binds through noncovalent bonds.Inhibition can be reversed by dialysis or petitive inhibitionnon-competitive inhibitionuncompetitive inhibition forms1)Competitive inhibition A competitive inhibitor typically has close structur
43、al similarities to the normal substrate for the enzyme.I t c o m p e t e s w i t h t h e substrate molecules for binding to the active site of the enzyme.Competitive inhibition At high substrate concentration,the effect of a competitive inhibitor can be overcome.v=Vmax SKm(1+SKiI)E+SE+P+IEIESKiCompe
44、titive inhibitioninhibitor No inhibitorLineweaver-Buck plotKm increasedVmax unchanged No inhibitorinhibitorvExample of competitive inhibitionClinical application of competitive inhibition(2)Noncompetitive inhibition A noncompetitive inhibitor binds at a site other than the active site of the enzyme
45、and decreases its catalytic rate by causing a conformational c h a n g e i n t h e t h r e e-dimensional shape of the enzyme.Noncompetitive inhibitionE+SESE+P+IESI+IEI+SE+SESE+P+IESI+IEI+SKiKi The inhibitor usually bears little or no structural resemblance to S.The ESI complex does not proceed to fo
46、rm product.The effect of a noncompetitive inhibitor cannot be overcome at high substrate concentrations.Noncompetitive inhibitioninhibitor No inhibitor)Ki(1V1S1)Ki(1VKV1mmmII+=Lineweaver-Buck plotKm unchangedVmax decreased(3)Uncompetitive inhibitionUncompetitive inhibitor The inhibitor bind to ES co
47、mplex and results in decrease of both ES and P(also free E).The ESI complex can not be reversed by increasing S.no inhibitorinhibitorUncompetitive inhibitionKiE+SESE+PESI+ILineweaver-Buck plot )KI(1V1S1VKv1imaxmaxm+=Km decreasedVmax decreasedInhibitor TypeBinding Site on EnzymeReactionCompetitive In
48、hibitorSpecifically at the catalytic site,where it competes with substrate for binding in a dynamic equilibrium-like process.Inhibition is reversible by substrate.Noncompetitive InhibitorBinds E or ES complex other than at the catalytic site.Substrate binding unaltered,but ESI complex cannot form pr
49、oducts.Inhibition cannot be reversed by substrate.Uncompetitive InhibitorBinds only to ES complexes at locations other than the catalytic site.Substrate binding modifies enzyme structure,making inhibitor-binding site available.Inhibition cannot be reversed by substrate.KiE+SESE+PESI+IE+SESE+P+IESI+I
50、EI+SE+SESE+P+IESI+IEI+SKiKiproperties No inhibitorsCompetitiveinhibitorsnoncompetitiveinhibitorsuncompetitiveinhibitorsComponent binding to IEE、ESESApparent KmKmunchangedVmax VmaxunchangedLineweaver-Burk plot intercept at x axis-1/KmunchangedIntercept at y axis1/VmaxunchangedslopeKm/VmaxunchangedTab
