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UVM BIOC 205 - Introduction to Enzymes and Kinetics

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Lecture 12: Enzymes & Kinetics IIntroduction to Enzymes and KineticsMargaret A. DaughertyFall 2003ENZYMES: Why, what, when, where, how?All but the who!What: proteins that exert kinetic control over biological reactionsWhy: We need to have chemical reactions occur on a biologicllyrelevant time scale (recall the example from lecture 1)When: At appropriate times during metabolic processes (brings usto the idea of biological regulation - lecture 16).Where: Where ever chemistry needs to be accomplished in the bodyHow: Through the bringing together of reactive groups on theenzyme with the substrates that need to have chemistry performedon them.BIOC 205THERMODYNAMIC POTENTIALITY: reaction is stronglyexergonic; but doesn’t occur under “normal” conditions. It needshelp….Enzymes provide the help -they lower the activationenergy necessary to have areaction go to completionBIOC 205What is an enzyme?General Properties• Mostly proteins, but some are actually RNAs• Biological catalysts– Higher reaction rates– Milder reaction conditions• Great reaction specificity• Capacity for regulation• Not changed or used up after a reaction• Nomenclature: frequently add -aseBIOC 205KEY FEATURES OF ENZYMESCATALYTIC POWER: ratio of catalyzed reaction rate touncatalyzed reaction rate; enzymes accelerate reactions asmuch as 1020; important to note that they do this underphysiological conditions (pH 7, 37C, H20)BIOC 205SPECIFICITY: Enzymes are selective about their substrates (alsocalled ligands, reactants) and the chemistry they carry out (activesites are specialized for both the reactant and the chemistry).There are no wasteful by-products.KEY FEATURES OF ENZYMESBIOC 205REGULATION: Enzymes should only function when needed. They areexquisitely regulated at the level of DNA, by interactions withinhibitors and activators, by product feed-back inhibition…….KEY FEATURES OF ENZYMESBIOC 205Glycogen Phosphorylase1). Response to fuel needs:High fuel state:Enzyme “off”high ATPhigh Glucosehigh G6PLow fuel state:Enzyme “on”high AMPlow ATP…2). Covalent modificationStress situation!Molecule on!Enzymes as Catalyststhe take home points• Enzymes DO NOT change the equilibriumconstant of a reaction• Enzymes DO NOT alter the amount of energyconsumed or liberated in a reaction (∆H);• Enzymes DO increase the rates of reactionsthat are otherwise impossible;• Enzymes DO decrease the activation energy(∆G‡);BIOC 205Enzymes• Enzymes are protein catalysts– Bind the substrates– Lower the activation energy– Directly promote the catalytic eventsCatalyst RateenhancementNon-enzymatic (metals) 102 - 104 foldEnzymatic up to 1020 foldHow much is 1020 fold?- with a catalyst, the reaction takes place in 1 sec- without a catalyst, 3 x 1012 years!BIOC 205Thermodynamics tells it willproceed in the direction of BTransition State and Free EnergyConsider a reversible reaction A<----->BBIOC 205Transition statetheory providesinformation on ∆G‡and says that ∆G1‡is smaller than∆G-1‡, thusreaction favorsformation of BTransition State and Free Energy∆G‡: rate is proportional to # of molecules that have this energyk= Ae-∆G‡/RT(Arrhenius Equation)∆G‡ = Free Energy ofActivation determinesthe rate of reactionBIOC 205Catalytic ReactionsCatalysts (e.g. enzymes) act by lowering the transitionstate free energy for the reaction being catalyzed.A BDo not raise energy of ABIOC 205Six Major Classes of Enzymes• Oxidoreductases: oxidation-reduction reactions• Transferases: transfer of functional groups• Hydrolases: cleavage of bonds by hydrolysis• Lyases: group elimination to form double bonds• Isomerases: isomerization (simplest)• Ligases: bond formation between 2 substratesBIOC 205Oxidoreductases• Oxidation-reduction reactions(addition or removal of hydrogenatoms from many chemicalsubstituents)• Example: dehydrogenasesOxidases, oxygenases, reductases, peroxidses & hydroxylasesBIOC 205Transferases• Transfer of functional groups betweendonor and acceptor molecules;• Example: kinasesGroups: amino, carboxyl, carbonyl, methyl, phosphoryl and acyl (RC=0)BIOC 205Hydrolases• Cleavage by hydrolysis reactions (addingH2O across a bond);• Example: the proteases(esterases, phosphatases & peptidases)BIOC 205Lyases• Group elimination or addition to doublebonds;• Frequently H2O, NH3 or CO2;• Example: pyruvate decarboxylate(hydratases, dehydratases, deaminases, synthases)BIOC 205Isomerases• Isomerization reactions (intramolecularrearrangements);• Example: alanine racemaseEpimerases: catalyze interconversion of asymmetric carbon atomsMutases: catalyze intramolecular transfer of functional groupsBIOC 205Ligases• Bond formation by condensation of twogroups coupled to ATP hydrolysis;• Example: polymerases(synthetases, carboxylases)BIOC 205Some examples: To what class of anenzyme do the following enzymes belong?BIOC 205COENZYMES: ENZYMES NEED “HELP” Recall our definitions of apoprotein,prosthetic group, holoenzyme)CHEMICAL KINETICSFIRST ORDER REACTIONS & THE RATE CONSTANTA Bk1k-1k1 = rate constant forthe forward reactionk-1 = rate constant forthe reverse reaction (units = sec-1)The rate law:V = d[B]/dt or -d[A]/dtV = -d[A]/dt = k[A]orAt equilibriumV = k1[A] - k-1[B] = 0k1k-1=[B]eq[A]eq=KeqCHEMICAL KINETICSSECOND ORDER REACTIONS & THE RATE CONSTANTAt equilibriumV = k2[A]2 - k-2[A2] = 0k2k-2=[A2]eq[A]2eq=KeqV = -d[A]2/dt = k2[A]2 Rate Law:SIMPLE EXAMPLE2A A2k2k-2k2 = (moles/L)-1sec -1A + B C + Dk2k-2BIOC 205Enzymes and EquilibriumkfA BkrK = = = = 100kfkr[B][A]10-410-6Therefore, at equilibrium the [B] is 100 times [A]Note: Enzymes accelerate the attainment of equilibria, but they DO NOT shift their positions!kf = 10-4 s-1kr = 10-6 s-1BIOC 205Enzyme Reactions• Physically interact with their substrates toeffect catalysis;• Substrates bind to the enzyme’s activesiteE + S ES ES* EP E + PE = enzymeES = enzyme-substrate complexES* = enzyme/transition state complexEP = enzyme product complexP = productReview1). Enzymes are protein catalysts that speed up biological reactions by as muchas 1020.2). Enzymes work by reducing the ∆G‡, not by altering the equilibrium constant.3). ∆G‡, is the additional energy that substrates have to have above and beyondtheir intrinsic energy to reach the transition state. By reducing the ∆G‡, thereare molecules that can reach the transition


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UVM BIOC 205 - Introduction to Enzymes and Kinetics

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