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    Enzyme Inhibition

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    Jane Docdoc
    This document describes an experiment analyzing enzyme kinetics under two conditions. Condition A measured initial reaction rates (V0) at various substrate concentrations without inhibitor. Condition B measured V0 with inhibitor present. The results were used to: 1) Calculate Vmax, Km, and kcat for condition A. 2) Determine apparent Vmax and Km for condition B, indicating competitive inhibition. 3) Calculate the inhibition constant KI, showing the inhibitor binds about twice as tightly as the substrate.

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    Enzyme Inhibition

    Uploaded by

    Jane Docdoc
    270 views7 pages
    This document describes an experiment analyzing enzyme kinetics under two conditions. Condition A measured initial reaction rates (V0) at various substrate concentrations without inhibitor. Condition B measured V0 with inhibitor present. The results were used to: 1) Calculate Vmax, Km, and kcat for condition A. 2) Determine apparent Vmax and Km for condition B, indicating competitive inhibition. 3) Calculate the inhibition constant KI, showing the inhibitor binds about twice as tightly as the substrate.

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    biochem engg

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    This document describes an experiment analyzing enzyme kinetics under two conditions. Condition A measured initial reaction rates (V0) at various substrate concentrations without inhibitor. Condition B measured V0 with inhibitor present. The results were used to: 1) Calculate Vmax, Km, and kcat for condition A. 2) Determine apparent Vmax and Km for condition B, indicating competitive inhibition. 3) Calculate the inhibition constant KI, showing the inhibitor binds about twice as tightly as the substrate.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    270 views7 pages

    Enzyme Inhibition

    Uploaded by

    Jane Docdoc
    This document describes an experiment analyzing enzyme kinetics under two conditions. Condition A measured initial reaction rates (V0) at various substrate concentrations without inhibitor. Condition B measured V0 with inhibitor present. The results were used to: 1) Calculate Vmax, Km, and kcat for condition A. 2) Determine apparent Vmax and Km for condition B, indicating competitive inhibition. 3) Calculate the inhibition constant KI, showing the inhibitor binds about twice as tightly as the substrate.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 7

    PROBLEM:

    The table gives enzyme-catalyzed reaction rates (initial rate, V0) measured at various
    substrate concentrations in solutions with [E] = 1.2 x 10-4 mmol/L.

    1. Use the data from Experiment 1 to calculate Vmax, Km, and kcat for this enzyme-catalyzed
    reaction.
    2.Use the data from Experiment 2 to determine the apparent Vmax and Km in the presence of the
    inhibitor.
    3.From this information, determine the type of inhibition (competitive, noncompetitive or
    uncompetitive) and calculate the dissociation constant KI for the inhibitor.
    Experiment 1:
    •Vmax is the inverse of the (1/V0) -intercept on the Lineweaver-Burke plot:

    Vmax = 1/(4.6155 [L-min/mmol S]) = 0.217 mmol S/L-min

    •The slope of the Lineweaver-Burke plot is equal to Km/Vmax, so Km = slope x Vmax:

    Km = (0.378 [L-min/mmol S]/[L/mmol S]) x (0.217 [mmol S/L-min]) = 0.0820 mmol


    S/L

    •Vmax = kcat x [E]total so kcat = Vmax/[E]total

    kcat = (0.217 [mmol S/L-min]/(1.2 x 10-4 [mmol E/L] = 1.81 x 103 [(mmol S/mmol
    E)/min] x (1 min/60 sec)

    kcat = 30.1 sec-1


    Experiment 2:
    Using the same methods as in experiment 1,
    •Vmax(apparent) = 0.183 mmol S/L.min Inhibition is competitive, but note that
    with realistic data like this, there is some
    uncertainty in this conclusion.
    •Km(apparent) = 0.152 mmol S/L
    inhibition is competitive because the
    KI = Km[I]/(Kmapp - Km) inhibitor raises Km by almost 85%

    KI = (0.0820 [mmol S/L] x 0.033 [mmol I/L])/ lowers Vmax by only 16%.
    (0.152 [mmol S/L] - 0.0820 [mmol S/L]) Given the poor data (look at the
    R2 values), the change is Vmax is
    KI = 0.039 mM probably not significant.

    Compare KI with Km (not Kmapp).


    The inhibitor binds about twice as tightly as
    the substrate.
    ENZYME KINETICS – SAMPLE PROBLEM An enzymatic
    assay was carried under two different sets of conditions
    out using a pure substrate S. The results are tabulated
    below. [S]/ Vo 10-5 M Condition A Condition B 1.5 0.21
    0.08 2.0 0.25 0.1 3.0 0.28 0.12 4.0 0.33 0.13 8.0 0.44 0.16
    16.0 0.40 0.18 a. Plot the data using the Lineweaver-
    Burke plot b. Calculate the values of V max and K m for
    both sets of conditions c. Suggest possible reasons why
    the two sets of results might be different. ENZYME KINETICS
    – SAMPLE PROBLEM An enzymatic assay was carried under two
    different sets of conditions out using a pure substrate S. The
    results are tabulated below. [S]/ Vo 10-5 M Condition A
    Condition B 1.5 0.21 0.08 2.0 0.25 0.1 3.0 0.28 0.12 4.0 0.33
    0.13 8.0 0.44 0.16 16.0 0.40 0.18 a. Plot the data using the
    Lineweaver-Burke plot b. Calculate the values of V max and K m
    for both sets of conditions c. Suggest possible reasons why the
    two sets of results might be different.

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