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General Features of Enzymes. Most biological reactions are catalyzed by enzymes Most enzymes are proteins Highly specific (in reaction & reactants ) Involvement of cofactor or coenzyme in some enzymes (prosthetic groups, holoenzyme, apoenzyme) Activity regulated through

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General features of enzymes
General Features of Enzymes

  • Most biological reactions are catalyzed by enzymes

  • Most enzymes are proteins

  • Highly specific (in reaction & reactants)

  • Involvement of cofactor or coenzyme in some enzymes

    (prosthetic groups, holoenzyme, apoenzyme)

  • Activity regulated through

    • Feedback inhibition

    • Regulatory proteins (e.g. calmodulin)

    • Covalent modification (e.g. phosphorylation)

    • Precursor to mature form transition

      (proteolytic activation)


How enzymes work
How Enzymes Work

  • Substrate binding is the first step of enzymatic catalysis

    • Substrate

    • Active site

      • Binds substrate (by multiple weak interactions)

      • A 3-dimensional entity complementary to substrate

      • Contains catalytic residues

      • Size and location: Small; located at clefts or crevices

      • Source of binding specificity



Enzymes Accelerate Reaction Rate

How?

  • Enzymes accelerate reaction ratebutdo not alter equilibrium!

    • Rate of reaction= (Ae-G‡/RT)[S]

    • Accelerate reaction rate by stabilizing transition states (G‡)

    • Essence of catalysis: specific binding of the transition state


Michaelis menten model accounts for kinetic properties of many enzyme

k1

k2

Michaelis-Menten ModelAccounts for Kinetic Properties of many Enzyme

k3

  • Kinetic properties of many enzymes (V vs. [S] plot)

  • Michaelis-Menten Model

    E + S ES E + P

    • Purpose: using the model to derive an expression relating

      rate of reaction to [E] and [S] and k1, k2, and k3

    • Assumption #1: no product reverts to initial substrate (initial state)

    • Assumption #2: steady state ([ES] is constant)

      • k1[E][S]=k2[ES]+k3[ES], so [ES] = [E][S]/KM ; KM =(k2+k3)/k1

      • [E] = [ET] - [ES]; [S] = [ST] - [ES] - [P]

      • work under the following condition: [ET] << [ST] ; and at initial time, so [P] is negligible, and so [S] = [ST]  [ES] = [ET] [S]/(KM + [S])

        so, V = k3 [ES] = k3[ET] [S]/(KM + [S]) = Vmax [S]/(KM + [S])


  • Michaelie-Menten equations

    explains the kinetic trend

    seen for many enzymes

    V = Vmax [S]/(KM + [S]):

    • When [S] << KM, V = Vmax [S]/KM ,

      V is directly proportional to [S]

    • When [S] >> KM , V = Vmax ,

      rate is maximal, independent of [S]

    • When [S] = KM, V = (1/2) Vmax,

      so, KM = [S] when V is 1/2 Vmax


  • Determine KM and Vmax

    • Experimental Procedure

      • Set up several reactions with fixed [ET] but increasing [ST]

      • Experimentally determine V at various [ST] (simplified as [S];

        V is initial velocity so [P] is negligible)

    • Data Analysis

      • Using Michaelis-Menten Equation:

        V = Vmax [S]/(KM + [S])

        • Plot V vs. [S]; computer curve fitting to find KM and Vmax

      • Lineweaver-Burk Plot

        1/V = 1/Vmax + (KM/Vmax) 1/[S]

        • Plot 1/V vs. 1/[S]

        • Y intercept = 1/Vmax; X intercept = -1/KM


Kinetic Perfection in Enzymatic Catalysis

  • For Enzymes that Obey Michaelis-Menten Model

    • When all enzyme molecules are saturated with substrate

      • V = Vmax = k3 [ET], rate constant is k3 (= kcat)

    • When [S] << KM and so most of the active sites are unoccupied

      • V = k3 [ES]= k3 [E][S]/KM

        as [S] << KM, so [E]  [ET], so V = k3 [ET][S]/KM = (k3/KM)[ET][S]

        so V depends on k3 / KM: k3 / KM= k3 k1 / (k2 + k3) < k1

        k1 cannot be faster than diffusion controlled encounter of

        an enzyme and its substrate, which is108 to 109 M-1 s-1

        So, the upper limit of k3 / KM is 108 to 109 M-1 s-1.

  • For Enzymes that Do not Obey Michaelis-Menten Model

    • When all E are saturated with S, rate depends on k cat; kcat  k3

    • When not all E are saturated with S, rate depends on k cat / KM

  • Some enzymes having k3/KM of 108 - 109 M-1 s-1 reached kinetic perfection! Their catalytic velocity is limited by the rate at which they encounter substrate in the solution.


Enzyme Inhibition

  • Irreversible Inhibition

    • Inhibitor destroys a functional group on the enzyme

    • Or inhibitor binds to the enzyme very tightly (covalently or noncovalently)  dissociates very slowly from enzyme

  • Reversible Inhibition


  • Reversible Inhibition

    • Inhibitor binds and dissociate rapidly from the enzyme

    • Competitive inhibitor

      • Inhibitor binds at active site; compete for binding with substrate; exist as either ES or EI; no ESI

      • Inhibitor structure resembles that of substrate

      • Overcome competitive inhibition by increasing [S]

    • Noncompetitive inhibitor

      • Inhibitor binds at a site other than active site

      • Binding of noncompetitive inhibitor decreases turnover number (reduces k3)


Kinetics of Enzyme Inhibition

  • Assume the enzyme exhibits Michaelis-Menten Kinetics

    • Set up enzymatic reactions with fixed [ET] but increasing [ST]

    • One set without inhibitor and another set with inhibitor

    • Plot 1/V vs. 1/[S] (Lineweaver-Burk Plot)


Kinetics of Enzyme Inhibition

  • Competitive Inhibition

    • The two lines on the plot have the same Y intercept (Same V max)

    • KM and KIM are different : KIM = KM (1 + [I]/KI)

      KI = [E][I]/[EI] (for E + I EI)

    • 1/V = 1/Vmax + KM/Vmax (1 + [I]/KI) (1/[S])

    • KM and KIM can be determined from the Lineweaver-Burk plot

    • KM’ = KM (1 + [I]/KI) allows the determination of KI

    • Inhibition can be overcome

      by increasing [S]


Kinetics of Enzyme Inhibition

  • Noncompetitive Inhibition

    • Same KM in the presence and absence of Inhibitor

    • Smaller V max in the presence of Inhibitor

    • VI max = V max /(1 + [I]/KI)

    • VI max and V max can be determined from the Lineweaver-Burk plot

    • VI max = V max /(1 + [I]/KI)

      allows the determination of KI

    • Cannot be overcome

      by increasing [S]


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