2The enzyme Aequorin catalalyzes the oxidation of this compd in presence of calcium to release CO2 and light
3Properties of EnzymesEnzymes are protein catalysts. Increase rate of chemical reactionNOT CONSUMED.Show high catalytic efficiency…enzyme catalyzed reactions from 103 to 106 times faster.Highly specific, catalyze only one type of chemical reactionSome have non-protein cofactor needed for enzymatic activity.Cofactors commonly metal ions like Zn2+ and Fe2+, derivatives of vitamins, NAD+ , FAD, and Coenzyme A.
4Terminology Holoenzyme = enzyme (protein) + cofactor (non protein) absence of cofactor, no activityApoenzyme: protein portion of holoenzymeZymogen (Proenzyme): catalytically inactive enzyme complex (Ex: Chymotrypsinogen, pepsin)Prosthetic group: tightly bound cofactor that does not dissociate from enzyme (Ex: Biotin).
5Enzymes Powerful, Highly Specific Catalysts Accelerate reactions by six orders of magnitude or more.Carbonic anhydrasecatalyzes hydration of carbon dioxideOne of fastest enzymes knownCatalyzed reaction is 107 times as fast as uncatalyzed one
6Enzymes Powerful, Highly Specific Catalysts Precise interaction of substrate with enzymeThree dimensional structure of enzymeSpecific in terms ofReactionSubstrateCatalyzes single chemical reaction or a set of closely related reactionsEg. Proteases: catalyze hydrolysis of peptide bonds.Most proteolytic enzymes also catalyze a different but related reaction in vitrohydrolysis of an ester bond
7Enzymes Are Powerful and Highly Specific Catalysts Substrate specificityProteolytic enzymes differ markedly in their substrate specificityPapainWide substrate specificityCleaves any peptide bondTrypsinspecificCleaves only on the C terminus of Lysine and Arginine residuesThrombinVery SpecificCatalyzes hydrolysis of Arg-Gly bonds in a particular sequence only
8CofactorsCatalytic activity of enzymes depends on presence of small molecules, CofactorsApoenzyme + cofactor = holoenzymeCofactorsMetalsSmall organic molecules: coenzymesDerived from vitaminsTightly bound termed prosthetic groupsLoosely bound like co-substrates
10Free energy Useful Thermodynamic Function to Understand Enzymes Key activity in living systems is ability to convert one form of energy into anotherPhotosynthesisLight energy converted into chemical energyEnzymes play vital roles in energy transformationPlay fundamental role in photosynthesis and cellular respirationOther enzymes use chemical bond energy of ATP in diverse waysMyosin converts bond energy of ATP into mechanical energy of contracting muscles
11Free energy Useful Thermodynamic Function to Understand Enzymes Thermodynamic properties of reactionFree energy difference between reactants and productsDetermines if reaction occurs or notEnergy to initiate conversion of reactants into productsDetermines rate of reactionAffected by enzymes
12Bioenergetics Study of energy changes in biochemical reactions Why some reactions occur while others do not.Bioenergetics deals with energy:ReleaseStorageUse in biological systemsNonbiological reactions utilize heat energy.Biological systems isothermic, use chemical energy.
13Free energy Useful Thermodynamic Function to Understand Enzymes Free energy change (ΔG) of chemical process is measure of energy available to do work.ΔG = Gproducts – GreactantsIf ΔG <0 , forward reaction favored.Products more stable than reactants. As reaction proceeds, energy released that can do work.If ΔG = 0, reactants and products at equilibriumIf ΔG >0, reactants at lower energy than products; energy needed for reaction.ΔG is independent of the path of the transformationΔG for oxidation of glucose the same whether combustion or enzyme catalyzed reactionsΔG provides no information on rate of reaction. Rate depends on free energy of activation, unrelated to ΔG of reaction
14Free energy Useful Thermodynamic Function to Understand Enzymes Standard Free Energy Change is related to Equilibrium ConstantΔGo’ is directly dependent on the equilibrium constant K’eq.For a reaction:A B C + DΔG = ΔGo’ + RT. ln [C] [D]/[A] [B]ΔG depends on the nature of the reactants (expressed in the ΔGo’ term) as well as the concentration of the reactant and productsAt equilibrium, ΔG = 0, and K’eq = [C] [D]/[A] [B]ΔGo’ = - RT.ln K’eq or ΔGo’ = RT log K’eqsince R= x 10-3 kJ.mol-1.deg-1 and T = 298K,ΔGo’ = log K’eq or K’eq = 10 –ΔGo’ /5.71The free energy change of the forward reaction is equal in magnitude but opposite in sign to that of the backward reaction.A B -5 kcalB A +5 kcal
15Important FactsCriterion for reaction under specified conditions depends on DG not DG0. Knowing concentrations of reactants allows determination!Standard free energy changes additive in any sequence of consecutive reaction:As long as the sum of DGs of individual reactions is negative, the pathway can potentially proceed as writtenEven if some of the individual component reactions of the pathway have +DGs.
16Isomerization of DHAP to GAP At 250C at equilibrium[GAP]/[DHAP] =ΔGo’ = - RT.ln K’eq= X 10-3 X 298 X ln (0.0475)= 7.53 kJ/molReaction is endergonic and will not convert spontaneouslyFor initial concentrationFor [DHAP]/[GAP] = 0.015ΔG = ΔGo’ + RT.ln K’eq= 7.53kJ/mol kJ/mol = -2.89kJ/molΔG indicates it is exergonic and spontaneousCriterion of spontaneity for a reaction is ΔG; not ΔGo’Reactions not spontaneous based on ΔG can be spontaneous byadjusting concentrations of reactants and products.
17Additive property of free energy change Two or more reactions coupled when products of one reaction are reactants of next reaction.Multiple reactions coupled, become a pathway.Pathway must satisfy minimally two criteria:The individual reactions must be specific, yielding only one particular product or set of products.The entire set of reactions in a pathway must be thermodynamically favoredAn important thermodynamic fact: the overall free energy change for a chemically coupled series of reactions is equal to the sum of the free-energy changes of the individual stepsAs long as the sum of ΔG’s of the individual reactions is negative the pathway is thermodynamically favorable.A B + C G0’ = + 5 kcal mol-1B D G0’ = - 8 kcal mol-1*******************************A C + D G0’ = - 3 kcal mol-1
18Enzymes Only alter reaction rate Cannot alter reaction equilibrium Eg. Conversion of S to PkF = 10-4 s-1 and kR = 10-6 s-1K = [P]/[S]=kF/kR = 100Equilibrium concentration of P is 100 times that of substrate, irrespective of enzymeMight take a very long time to achieve this equilibrium in absence of enzymeEquilibrium attainted rapidly in presence of suitable enzymeThus, enzymes accelerate attainment of equilibrium but do not shift positions.
19How Enzymes WorkAccelerate reactions by facilitating formation of the transition stateChemical reaction of substrate (S) to form product (P) goes through transition state (X‡)S X‡ PTransition state (X‡):Top of the energy hill;Highest free energy, least stableMay go to S or P.Not reaction intermediateBond breakage, bond formation, charge developments take place.Difference in free energy between transition state and substrate called “Gibbs free energy of activation” or “activation energy”G‡ = Gx‡ - GsV (reaction rate) proportional to G‡ G‡ does not enter G calculation as energy required to generate this is released during product formation.This is how enzymes alter reaction rate without altering G of reactionEnzymes function to lower activation energy or facilitates formation of transition state
20How Enzymes Work Enzymes form ES complex. Existence of ES complexes shown in many ways:At constant concentration of enzyme, reaction rate increases with increase in [S] until a Vmax is reached.Maximal velocity suggests formation of ES complexX-ray crystallographyImages of substrate analogs bound to active sitesSpectroscopic characteristics of many enzymes and substrate change upon formation of ES complex.
21Active SiteRegion that binds substrate and possesses catalytic residues that participate in reaction mechanism.Interaction of enzyme and substrate at active site promotes formation of transition state.Region that lowers activation energy of a reaction, thus speeds up reaction
22Active SiteAlthough structure, specificity, or mode of catalysis of enzymes differ, their active sites share common featuresThree dimensional cleft, or crevice, formed by groups from different parts of amino acid sequenceSmall part of total volume of enzymeEnzymes large three-dimensional structuresServe as scaffold to create 3D active siteConstitute regulatory sitesInteraction with other proteinsSubstrate channelsUnique MicroenvironmentsWater usually excluded unless a reactantNon polar environment enhances binding of substrates as well as catalysis.Polar residues acquire special properties essential for substrate binding and catalysis in microenvironment.
25Active SiteSubstrates bound to enzymes by multiple weak reversible interactionsElectrostaticHydrogen bondsDirectional character results in high degree of specificityVan der Waals forcesBecomes significant when numerous substrate atoms come close to many enzyme atoms which requires enzyme and substrate to have complementary shapes.
26The specificity of binding depends on the precisely defined arrangement of atoms in an active site. Since enzymes and substrates interact by means of short range forces that require close contact, a substrate must have a matching shape to fit into the site.Emil Fisher’s Lock and Key model (1890)Enzymes are flexible and shape of the active site can be markedly modified by the binding of substrateDaniel Koshland Jr.’s dynamic induced fit model (1958)Active site assumes a shape that is complementary to that of the substrate only after the substrate has been bound.
27Induced-Fit ModelIn absence of substrate, catalytic and substrate binding groups are far from each other.In presence of substrate, conformational change occurs in the enzyme… results inAligning groups correctly for substrate binding and catalysisSubstrate analogs cause some, not all correct conformational changes.Active site has shape complementary to that of substrate only after substrate is bound.
28Enzyme-Substrate binding energy Binding energy: Free energy released upon multiple weak interactions between enzyme and substrate.Maximized when right substrate interacts to form interactionsAccounts for substrate specificityMaximal energy released when enzyme facilitates formation of transition stateBinding energy lowers activation energyTransition state forms S or P depending reaction ΔG
29Definition of TermsSubstrate: substance acted upon by an enzyme. Activity: amount of substrate converted to product by enzyme per unit time (M/min). Specific activity: activity per quantity of protein (M/min/mg protein). International unit: quantity of enzyme needed to transform 1.0 micromole of substrate to product per minute at 30C and optimal pH.
30The Michaelis-Menten Equation Describes the Kinetic Properties of Many Enzymes
35Meaning of KM Two meanings: Measure of strength of ES complex Substrate concentration when half the active sites filledDissociation constant of ES complex when k2 is smallMeasure of strength of ES complexHigh KM weak bindingLow KM strong binding
38Importance of KM CH3CH2OH + NAD+ CH3CHO + H+ + NADH Some individuals sensitive to ethanol, exhibit facial flushing, rapid heart rate, after ingesting alcohol. In the liver:AlcoholdehydrogenaseCH3CH2OH + NAD+ CH3CHO + H+ + NADHAldehydeCH3CHO + NAD+ + H2O CH3COO- + NADH + 2H+
39Most people have 2 forms of acetaldehyde dehydrogenase: low Km, mitochondrial formhigh Km, cytosolic formIn susceptible persons:the mitochondrial enzyme less active because single amino acid substituted andacetaldehyde processed only by cytosolic enzyme.Cytosolic enzyme has high Km,less acetaldehyde converted into acetate,increased acetaldehyde goes to blood, andsymptoms appear.
40kcatTurnover number:Number of S molecules converted into product by enzyme molecule in a unit time when the enzyme fully saturated with substrate.It is equal to the k3.k3= Vmax/ET
42kcat/KM kcat/ KM can be used as a measure of catalytic efficiency. When the [S]>>>>KM, the rate of catalysis is equal to kcat.But, under physiological conditions, the [S]/KM is equal to (meaning [S]<<<<KM)The enzymatic rate is less than kcat because enzymes active sites are unoccupied.kcat/ KM can be used as a measure of catalytic efficiency.By using kcat/ KM, we can compare an enzyme’s preference for different substrates.
44Most biochemical reactions include multiple substrates. Multiple substrate reactions can be divided into 2 classes:Sequential displacementDouble displacementLactate dehydrogenase enzyme: ordered sequential (NADH must bind first and lactate released first)
45The enzyme (LDH) exists as a ternary complex Sequential displacement (ordered)The enzyme (LDH) exists as a ternary complex
46Sequential displacement (random) The order of addition of substrates and release of products is random! Creatine kinase is the enzyme.
50Allosteric enzymes Do not obey M-M model! Multiple subunits, multiple active sites.Display sigmoidal plots.Binding of S to one active site affects properties of other active sites in same enzyme molecule.
54Allosteric activators and inhibitors shift the curve in different directions (fES: fraction of sites filled)
55Enzymes inhibited by specific molecules Important because:Major control mechanismMany drugs and toxic agents act this way.2 kinds of enzyme activity:ReversiblecompetitivenoncompetitiveIrreversibleI dissociates very slowly from the enzyme because I binds to the enzyme covalently. (Ex.: nerve gases)
67Competitive inhibitors Resemble substrate, bind to active site of enzymeMethotrexate is structural analog of tetrahydrofolate(coenzyme of dihydrofolate reductase that is involved in purine and prymidine synthesis)Diminish rate of catalysis by reducing proportion of enzyme molecules bound to substrateCompetitive inhibitors are used suchmmmmmmmm
69Irreversible inhibitors used to map active site Functional groups important for enzymes activity.How can we be certain about functional groups?X-ray crystallographic of enzyme bound to its substrateIrreversible inhibitors provide alternative approach
70Three groups of irreversible inhibitors Group specificReacts with R groups of amino acidsDIPF(diisopropylphosphofluoridate)IodoacetamideSubstrate analogMolecules structurally similar to S for enzyme that covalently modifies active siteTPCK (tosyl-L-Phe chloromethyl ketone)3 bromoacetolSuicide inhibitorsEnzyme participates in its own irreversible inhibition
84Penicillin irreversibly inactivates key enzyme in bacterial cell wall synthesis Cell wall made of macromolecule called “peptidoglycan”, linear polysaccaride chains crosslinked by short peptides.Penicillin blocks last step of cell wall synthesis, i.e., crosslinking of different peptidoglycan strands.Penicillin inhibits crosslinking action of “glycopeptide transpeptidase” enzyme.Penicillin fits in active site of enzyme because looks like D-Ala-D-Ala!