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LECTURE 4: Principles of Enzyme Catalysis Reading: Berg, Tymoczko & Stryer: Chapter 8

LECTURE 4: Principles of Enzyme Catalysis Reading: Berg, Tymoczko & Stryer: Chapter 8. An ENZYME is a biomolecular catalyst that accelerates the rate of a specific reaction Enzymes DO NOT make a chemical reaction more energetically favorable;

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LECTURE 4: Principles of Enzyme Catalysis Reading: Berg, Tymoczko & Stryer: Chapter 8

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  1. LECTURE 4: Principles of Enzyme Catalysis Reading: Berg, Tymoczko & Stryer: Chapter 8 An ENZYME is a biomolecular catalyst that accelerates the rate of a specific reaction Enzymes DO NOT make a chemical reaction more energetically favorable; They only ACCELERATE the rate of the reaction towards its energetic equilibrium Enzymes work by stabilizing chemical transition states, the high-energy intermediates that normally act as a barrier to spontaneous reaction Most enzymes are FOLDED PROTEINS: proteins have the ability to fold into scaffolds with binding surfaces for substrates that position the substrates for chemical reaction A few enzymes are RNA molecules! RNAs also have ability to adopt tertiary structures. Some RNAs (called RIBOZYMES) act as enzymes catalyzing their own site-specific cleavage or that of other RNA molecules As catalysts, enzymes are NOT CONSUMED during reactions. S + E -----> ES -------> P + E In some cases, enyzmes are chemically modified during catalysis, but return to their original form after reaction cycle to allow further catalysis of substrate S + E -----> E*S -------> P + E

  2. Proteases Are Examples of Enzymes That Catalyze An Energetically Favored Process Fig 8.0new (peptide hydryolys) Peptide hydrolysis is an energetically favorable process, but normally occurs very slowly. PROTEASES are enzymes that catalyze peptide hydrolysis. Some proteases are rather NONSELECTIVE (e.g., papain) Other proteases are VERY SELECTIVE (e.g., trypsin, thrombin, fibrin) Fig 8.1new

  3. Some Enzymes Employ Cofactors Some enzymes use cofactors as part of the active site in enzymatic catalysis APOENZYME + COFACTOR --------> HOLOENZYME Many cofactors cannot be synthesized by humans, and must be obtained through diet as vitamins and minerals Tab8.2new

  4. Free Energy of Biochemical Reactions For reaction A + B C + D DG is the differential in free energy between the products vs. reactants If DG < 0, reaction is energetically favorable I.e., reactants will convert to products as system moves to equilibrium If DG = 0, reaction is already at equilibrium I.e., there will be no NET conversion of reactants to products If DG > 0, reaction is disfavored I.e., products will convert to reactants as system moves to equilibrium; the reverse reaction is favored Free energy DG is usually expressed in units kcal/mol or kcal x mol-1

  5. [C] [D] DG = DG o + RT ln [A] [B] [C]eq[D]eq [A]eq[B]eq Standard Free Energy is Related to Equilibrium Constant For reaction A + B C + D DG is free energy change when reactant and product concentrations are [A],[B],[C],[D] DGo is free energy change when reactant and product concentrations are each 1M WHAT DOES THIS MEAN? DGo is measure of whether reactants or products are favored if all components are at same concentration The actual concentration of reactants and products impacts on DG; even if DGo is unfavorable, high ratio of reactants to products can give favorable DG DGo can be related to the equilibrium constant, Keq, of the reaction DG o = - RT lnKeq = - 2.3RT log10Keq Keq = Since

  6. DG o = - RT lnKeq = - 2.3RT log10Keq Tab8.3new

  7. Enzymes Accelerate Rate Constant Without Altering Equilibrium Constant S P Keq = kf / kr Without enzyme = S P Keq = kf / kr With enzyme Fig8.2new

  8. Enzymes Stabilize Reaction Transition State(s) Fig8.3new

  9. Properties of Enzyme Active Sites Active site consists of atoms on residue side chains that are brought together by the fold Most of the enzyme structure is a scaffold to precisely position active site residues Active site uses range of noncovalent bonding mechanisms to bind substrate By binding multiple substrates in a favorable interspatial relationship and/or by altering charge distribution (resonance) within substrates, DGtransition is much smaller than would be spontaneously Fig8.7new Fig8.8new

  10. = k2 kcat [S] Vo = VMAX [S] + KM The Michaelis-Menton Model of Enzyme Function k1 k2 E + S ES E + P k-1 k-2 k1 kcat At time=0, if [P]=0, then E + S ES E + P k-1 Vo = kcat [ES] [ES] determined by [E], [S], and rate constants THESE EQUATIONS CAN BE SOLVED TO EXPRESS THE REACTION RATE AS A FUNCTION OF THE SUBSTRATE CONCENTRATION [S] AND TWO INHERENT PROPERTIES OF THE ENZYME: KM AND kcat Michaelis-Menton Equation where KM = k-1 /k1 = [E][S]/[ES] and VMAX = kcat [E]

  11. [S] Vo = VMAX [S] + KM The Michaelis-Menton Equation: Meaning Behind The Terms No matter how large the substrate concentration, reaction rate can never exceed VMAX VMAX reflects the TURNOVER RATE of substrate molecules through the enzyme (kcat) and the enzyme concentration KM is the substrate concentration at which reaction rate is HALF MAXIMAL KM reflects the BINDING AFFINITY of the enzyme for the substrate; The higher the affinity, the smaller is Km By performing experiments to calculate Vo at different substrate concentrations, properties VMAX and Km can be calculated If the enzyme concentration is known, VMAX can be used to calculate kcat Fig8.12new

  12. [S] Vo = VMAX [S] + KM Lineweaver-Burk Plot Facilitates Calculation of KM and VMAX Fig8.13new By inverting equation, get: ( ) 1 1 KM 1 = + Vo VMAX VMAX [S] Tab8.5new

  13. Many Enzymatic Reactions Proceed Through Fixed Sequential Steps PyrLac.p223new TransAm.p224new Reaction Intermediate May Utilize Covalently Modified Enzyme or Cofactor

  14. Enzyme Inhibition Many small molecules can bind to enzymes and inhibit them. Inhibitors can be described as REVERSIBLE or IRREVERSIBLE. Inhibitors may be naturally occuring within the homologous organism or in a heterologous organism Other inhibitors are synthetic and have been developed as pharmaceuticals for research and clinical applications COMPETITIVE INHIBITORS act by occupying the enzyme active site in place of the substrate NONCOMPETITIVE INHIBITORS bind away from the active site, but their binding exerts allosteric effects that prevents bound substrate conversion to product Fig8.15Anew Fig8.15Bnew Fig8.15Dnew

  15. ( ) [ I ] 1 + Ki Kinetics of Competitive Inhibition [S] Vo = VMAX [S] + KM Ki reflects the AFFINITY OF INHIBITOR for the enzyme Inhibitor in effect raises the apparent KM term The potency of inhibitor determined by Ki Therefore, the amount of substrate needed for half-maximum rate is increased THERE IS NO EFFECT ON VMAX I.e., a competitive inhibitor can be overcome by sufficiently high substrate concentration Fig8.17new

  16. VMAX [S] Vo = x ( ) [ I ] [S] + KM 1 + Ki Kinetics of Noncompetitive Inhibition As before, Ki reflects the AFFINITY OF INHIBITOR for the enzyme Inhibitor reduces VMAX of the enzyme The potency of inhibitor determined by Ki The inhibitor does not affect KM , i.e., the binding of substrate to enzyme Fig8.19new

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