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  • Enzyme Kinetics - Inhibition

  • Types of Inhibition

    • Competitive Inhibition

    • Noncompetitive Inhibition

    • Uncompetitive Inhibition

    • Irreversible Inhibition

  • S

    I

    Enzyme

    Competitive Inhibition

    In competitive inhibition,

    the inhibitor competes

    with the substrate for the

    same binding site

  • Competitive Inhibition

    - Reaction Mechanism

    In competitive inhibition, the inhibitor binds only to the

    free enzyme, not to the ES

    complex

    E + S ES E + P

    EI

    + I

  • General Michaelis-Menten Equation

    This form of the Michaelis-Menten equation

    can be used to understand how each type of

    inhibitor affects the reaction rate curve

    v = [S]

    Km,app + [S]

    Vmax,app

  • In competitive inhibition, only the apparent Km

    is affected (Km,app> Km),

    The Vmax remains unchanged by the presence

    of the inhibitor.

  • Competitive inhibitors alter the

    apparent Km, not the Vmax

    Vmax

    Vmax 2

    Km Km,app

    [Substrate]

    R e a c ti o n R a te

    - Inhibitor

    + Inhibitor

    Vmax,app = Vmax

    Km,app > Km

  • The Lineweaver-Burk plot is

    diagnostic for competitive inhibition

    Slope = Km,app

    Vmax

    1 Vmax

    -1 Km,app

    1 [S]

    Increasing [I]

    1 v

    v =

    1

    Vmax

    Km,app

    Vmax

    1 +

    [S]

    1

  • Vmax

    Vmax 2

    Km Km,app

    [Substrate]

    R e a c ti o n R a te

    - Inhibitor

    + Inhibitor

    .

    S

    I

    Inhibitor

    competes with

    substrate,

    decreasing its

    apparent affinity:

    Km,app > Km

    Formation of EI

    complex shifts reaction

    to the left: Km,app > Km

    Km,app > Km Vmax,app = Vmax

    E + S ES E + P

    EI

    + I

    Formation of EI

    complex shifts reaction

    to the left: Km,app > Km

    Relating the Michaelis-Menten equation, the v vs. [S]

    plot, and the physical picture of competitive inhibition

  • Example - Competitive Inhibition

    Sulfanilamide is a competitive

    inhibitor of p-aminobenzoic

    acid. Sulfanilamides (also

    known as sulfa drugs,

    discovered in the 1930s)

    were the first effective

    systemic antibacterial

    agents.

    Because we do not make folic

    acid, sulfanilamides do not

    affect human cells.

    COOH

    NH2

    p-aminobenzoic acid

    folic acid

    SO2NH2

    NH2

    sulfanilamide

  • Practical case: Methanol poisoning

    A wealthy visitor is taken to

    the emergency room, where

    he is diagnosed with

    methanol poisoning. You

    are contacted by a 3rd year

    medical student and asked

    what to do? How would you

    suggest treating this

    patient?

  • Methanol (CH3OH) is metabolized to

    formaldehyde and formic acid by alcohol

    dehydrogenase. You advisethe third year

    student to get the patient very drunk.

    Since ethanol (CH3CH2OH) competes with

    methanol for the same binding site on

    alcohol dehydrogenase, it slows the

    metabolism of methanol, allowing the toxic

    metabolites to be disposed of before they

    build up to dangerous levels. By the way,

    the patient was very grateful and decided

    to leave all their worldly possessions to the

    hospital. Unfortunately, after being

    released from the hospital, he went to the

    casinos and lost everything he had.

  • S

    I

    I S

    S

    I

    I

    S

    Enzyme

    Enzyme

    Enzyme

    Enzyme

    Noncompetitive Inhibition

    the inhibitor does not interfere with substrate binding (and vice versa)

  • E + S ES E + P

    EI

    + I

    ESI

    + I

    S+

    Noncompetitive Inhibition -

    Reaction Mechanism

    In noncompetitive

    inhibition, the

    inhibitor binds

    enzyme

    irregardless of

    whether the

    substrate is bound

  • Noncompetitive inhibitors decrease

    the Vmax,app, but don’t affect the Km

    Vmax,app < Vmax

    Km,app = Km

    Vmax

    Vmax2 1

    2

    1 Vmax,app

    Km

    Km,app [Substrate]

    R e a c ti o n R a te

    - Inhibitor

    + Inhibitor

    Vmax,app

  • The inhibitor binds

    equally well to free

    enzyme and the ES

    complex, so it doesn’t

    alter apparent affinity

    of the enzyme for the

    substrate

    Why does Km,app = Km for

    noncompetitive inhibition?

    E + S ES E + P

    EI

    + I

    ESI

    + I

    S+

  • The Lineweaver-Burk plot is diagnostic

    for noncompetitive inhibition

    v =

    1

    Vmax,app

    Km

    Vmax,app

    1 +

    [S]

    1

    Slope = Vmax,app

    Km

    1 Vmax,app

    -1 Km

    1 [S]

    Increasing [I]

    1 v

  • Formation of EI

    complex shifts reaction

    to the left: Km,app > Km

    Km,app > Km Vmax,app = Vmax

    .

    S

    I

    I S

    S

    I

    I

    S

    Enzyme

    Enzyme

    Enzyme

    Enzyme

    .

    Vmax

    Vmax2 1

    2 1Vmax,app

    Km Km,app

    [Substrate]

    R e a c ti o n R a te

    - Inhibitor

    + Inhibitor

    Vmax,app

    Inhibitor doesn’t interfere with substrate binding,

    Km,app = Km

    E + S ES E + P

    EI

    + I

    ESI

    + I

    S+

    Even at high

    substrate levels,

    inhibitor still binds,

    [E]t < [ES]

    Vmax,app < Vmax

    Vmax,app < Vmax

    Km,app = Km

    Relating the Michaelis-Menten equation, the v vs. [S] plot,

    and the physical picture of noncompetitive inhibition

  • Noncompetitive inhibitors

    decrease the apparent Vmax, but

    do not alter the Km of the

    reaction

  • Example of noncompetitive inhibition:

    fructose 1,6-bisphosphatase inhibition by AMP

    fructose 1,6- bisphosphatase

    AMP

    OH2C

    H

    HO

    H

    CH2

    OH

    O O

    H

    OH

    P O- O

    -OP O- O

    -O

    fructose 1,6- bisphosphatase

    AMP

    fructose 1,6- diphosphate

    fructose 1,6- diphosphate

    AMPAMPAMPAMP

    fructose 1,6- bisphosphatase

    fructose 1,6- bisphosphatase

    OH2C

    H

    HO

    H

    CH2

    OH

    O O

    H

    OH

    P O- O

    -OP O- O

    -O

    fructose 1,6- bisphosphatase

    fructose 1,6- diphosphate

    fructose 1,6- diphosphate

    fructose 6- phosphate

    Pi

    E E.S E + P

    E.I E.S.I

  • Fructose 1,6-bisphosphatase is a key regulatory enzyme in the gluconeogenesis pathway. High amounts of AMP signal that ATP levels are low and gluconeogenesis should be shut down while glycolysis is turned on.

    High AMP levels inhibit fructose 1,6-bisphosphatase (shutting down gluconeogenesis) and activate phosphofructokinase (turning on glycolysis). Regulation of fructose 1,6-bisphosphatase and phosphofructokinase by AMP prevents a futile cycle in which glucose is simultaneously synthesized and broken down.

  • Uncompetitive Inhibition

    In uncompetitive

    inhibition, the

    inhibitor binds

    only to the ES

    complex

    .

    S

    I

    S

    Enzyme Enzyme

    I

    Enzyme

    S

    Enzyme

    I

  • Uncompetitive Inhibition -

    Reaction Mechanism

    In uncompetitive

    inhibition, the

    inhibitor binds only

    to the ES complex,

    it does not bind to

    the free enzyme

    E + S ES E + P

    ESI

    + I

  • Uncompetitive inhibitors decrease

    both the Vmax,app and the Km,app

    Vmax,app < Vmax

    Km,app < Km Notice that at low substrate

    concentrations,

    uncompetitive inhibitors

    have little effect on the

    reaction rate because the

    lower Km,app of the enzyme

    offsets the decreased Vmax,app

    Vmax

    Vmax2 1

    2

    1 Vmax,app

    KmKm,app [Substrate]

    R e a c ti o n R a te

    - Inhibitor

    + Inhibitor

    Vmax,app

  • Uncompetitive inhibitors decrease both the

    Vmax,app and the Km,app of the enzyme

    E + S ES E + P

    ESI

    + I

    Notice that

    uncompetitive inhibitors

    don’t bind to the free

    enzyme, so there is no

    EI complex in the

    reaction mechanism

  • The Lineweaver-Burk plot is

    diagnostic for uncompetiti

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