enzyme kinetics: study the rate of enzyme catalyzed reactions. - models for enzyme kinetics

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Enzyme Kinetics. Enzyme Kinetics: Study the rate of enzyme catalyzed reactions. - Models for enzyme kinetics - Michaelis-Menten kinetics - Inhibition kinetics - Effect of pH and Temperature. Enzyme Kinetics. - PowerPoint PPT Presentation

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  • Enzyme Kinetics: Study the rate of enzyme catalyzed reactions.

    - Models for enzyme kinetics- Michaelis-Menten kinetics- Inhibition kinetics- Effect of pH and TemperatureEnzyme Kinetics

  • Enzyme KineticsMichaelis-Menten kinetics or saturation kinetics which was first developed by V.C.R. Henri in 1902 and developed by L. Michaelis and M.L. Menten in 1913.

    This model is based on data from batch reactors with constant liquid volume.

    - Initial substrate, [S0] and enzyme [E0] concentrations are known.

    - An enzyme solution has a fixed number of active sites to which substrate can bind.

    - At high substrate concentrations, all these sites may be occupied by substrates or the enzyme is saturated.

  • Saturation Enzyme Kinetics

  • M-M Enzyme KineticsSaturation kinetics can be obtained from a simple reaction scheme that involves a reversible step for enzyme-substrate complex formation and a dissociation step of the ES complex.where the rate of product formation v (moles/l-s, g/l-min) isKi is the respective reaction rate constant.

  • Enzyme KineticsThe rate of variation of ES complex is

    Since the enzyme is not consumed, the conservation equation on the enzyme yields

  • Enzyme KineticsHow to use independent variable [S] to represent v?

  • At this point, an assumption is required to achieve an analytical solution.

    - The rapid equilibrium assumption Michaelis - Menten Approach.

    The quasi-steady-state assumption.Briggs and Haldane Approach. Enzyme Kinetics

  • Michaelis - Menten ApproachThe rapid equilibrium assumption:

    Assumes a rapid equilibrium between the enzyme and substrate to form an [ES] complex.

    E+SK-1K1

  • Michaelis - Menten ApproachThe equilibrium constant can be expressed by the following equation in a dilute system.

    E+SK-1K1

  • Michaelis - Menten ApproachThen rearrange the above equation,

    Substituting [E] in the above equation with enzyme mass conservation equation

    yields,

  • Michaelis - Menten Approach[ES] can be expressed in terms of [S],Then the rate of production formation v can be expressed in terms of [S],Where represents the maximum forward rate of reaction (e.g.moles/L-min).

  • Michaelis - Menten Approach- The prime reminds us that it was derived by assuming rapid equilibrium in the step of enzyme-substrate complex formation.

    Low value indicates high affinity of enzyme to the substrate.

    It corresponds to the substrate concentration, giving the half-maximal reaction velocity. Re-arrange the above equation,

    When is often called the Michaelis-Menten constant, mol/L, mg/L.

  • Michaelis - Menten ApproachVm is maximum forward velocity (e.g.mol/L-s)

    It changes with initial enzyme concentration.

    It is determined by the rate constant k2 of the product formation and the initial enzyme concentration.

    But it is not affected by the substrate concentration.The unit of k2 is determined by the unit of enzyme.

  • Briggs-Haldane ApproachThe quasi-steady-state assumption:A system (batch reactor) is used in which the initial substrate concentration [S0] greatly exceeds the initial enzyme concentration [E0]. since [E0] was small, d[ES]/dt 0

    - It is shown that in a closed system the quasi-steady-state hypothesis is valid after a brief transient if [S0]>> [E0].

  • The quasi-steady-state hypothesis is valid after a brief transient in a batch system if [S0]>> [E0].

  • Briggs-Haldane ApproachWith such assumption, the equation representing the accumulation of [ES] becomesSolving this algebraic equation yields

  • Briggs-Haldane ApproachSubstituting the enzyme mass conservation equationin the above equation yieldsUsing [S] to represent [ES] yields

  • Briggs-Haldane ApproachThen the product formation rate becomesThen,Where same as that for rapid equilibrium assumption.when K2
  • Comparison of the Two ApproachesMichaelis-Menten when k2
  • Experimentally Determining Rate Parameters for Michaelis-Menten Type Kinetics.

    To determine the rate parameters:- predict a specific enzyme catalysis system.- Design bioreactor

  • The determination of Vm and Km are typically obtained from initial-rate experiments.

    -A batch reactor is charged with known initial concentration of substrate [So] and enzyme [Eo] at specific conditions such as T, pH, and Ionic Strength.

    The product or substrate concentration is plotted against time.

    - The initial slope of this curve is estimated at t=0v=(d[P]/dt) , or = - (d[S]/dt) .

    This value v depends on the values of [E0] and [S0].

    - Many such experiments can be used to generate many pairs of V and [S] data, these data can be plotted as v-[S].

  • Initial slopes

    20102.5

    145.650.75

    930.45

    82.50.35

    72.30.3

    62.10.25

    51.90.2

    Slope

    Time (min)

    Substrate Concentration (M)

    Substrate Concernation versus Time

    New slopes

    20102.5

    145.650.75

    930.45

    82.50.35

    72.30.3

    62.10.25

    51.90.2

    new slope

    slope

    if sampled at 2 min interval, the initial rate of 20 M obtained by slope is changed.

    Time (min)

    Substrate Concentration (M)

    Substrate Concernation versus Time

    Sheet1

    Use initial rate experiemtnal data - Fumarase Kinetics

    t (time)S10 (M)S20 (M)S30 (M)

    020102.5

    5145.650.75

    10930.45

    1582.50.35

    2072.30.3

    2562.10.25

    3051.90.2

    To determine initial rates

    Slopedelta t (min)5Lineweaver-Burk PlotHanes-WoolfEadie-HofsteeNonlinear Regression

    S0S1V0 (M/s)1/V01/S0S0[S0]/V0V0/[S0]V0(S0-S)/t1/tln(S0-S)

    2014.31.140.87719298250.052017.54385964910.0571.141.140.0670945473

    105.650.871.14942528740.11011.49425287360.0870.870.870.1141859096

    6.73.20.71.42857142860.14925373136.79.57142857140.10447761190.70.70.1477913433

    52.050.591.69491525420.258.47457627120.1180.590.590.1783196239

    41.50.520.25480.1250.50.50.1961658506

    3.31.10.442.27272727270.3030303033.37.50.13333333330.440.440.2197224577

    2.90.950.392.56410256410.34482758622.97.43589743590.13448275860.390.390.2232008063

    2.50.750.352.85714285710.42.57.14285714290.140.350.350.2407945609

    y intercept=1/VmVm=1.7235436057Vm=1/slope1.6798252982y intercept Vm1.69y intercept =Vm/KmVm1.4092558984

    Slope-Km/VmKm=9.784901758y intercept=Km/VmKm=9.4021501764Km=-slope9.5Km=-1/slope4.5372050817

    Sheet1

    0

    0

    0

    0

    0

    0

    0

    0

    Slope = 1/Vm

    y intercept = Km/Vm

    x intercept = -Km

    [S0] (M)

    [S0]/V0 (min)

    Hanes-Woolf Plot

    y = 0.5953x + 5.5971

    Sheet2

    0

    0

    0

    0

    0

    0

    0

    0

    Slope = Km/Vm

    y intercept = 1/Vm

    x intercept = -1/Km

    1/[S0] (1/M)

    1/V0 (min/M)

    Lineweaver-Burk Plot

    y = 5.6772x + 0.5802

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    0

    y intercept = Vm/Km

    Slope =- 1/Km

    1/t(lnS0/S)

    (S0-S)/t

    Nonlinear Regression

    y = -0.2204x + 0.3106

    Sheet3

    0

    0

    0

    0

    0

    0

    0

    0

    y intercept = Vm

    Slope =-Km

    V0/[S0] (1/min)

    V0 (M/min)

    Eadie-Hofstee Plot

  • Lineweaver-Burk Plot (Double-Reciprocal Plot) Linearizing it in double-reciprocal form:

  • - a slope equals to Km/Vm y-intercept is 1/Vm. More often used as it shows the independent variable [S] and dependent variable v.-1/v approaches infinity as [S] decreases- gives undue weight to inaccurate measurement made at low concentration- give insufficient weight to more accurate measurements at high concentration.- Lineweaver-Burk plot (Double-reciprocal plot).

  • Eadie-Hofstee Plot- the slope is Km- y-axis intercept is Vm.Can be subject to large error since both coordinates contain dependent variable v, but there is less bias on points at low [s].

  • Hanes-Woolf (Langmuir) Plot- the slope is 1/Vmy-axis intercept is Km/Vmbetter fit: even weighting of the data

  • VmThe unit of Vm is the same as that of a reaction rate (moles/l-min, g/l-s)

    The dimension of K2 must reflect the units of [E0]-if the enzyme is highly purified, it may be possible to express [E0] in mol/l, g/l, then K2 in 1/time.

    - if the enzyme is crude, its concentration is in units. A unit is the amount of enzyme that gives a predetermined amount of catalytic activity under specific conditions.(Textbook, Bioprocessing Engineering, M. Shuler, p.66-67)

    If Vm is in mmol/ml-min, [E0] is in units/ml, then K2 should be in mmol/unit-min

  • Enzyme ActivitySpecific Activity is the number of units of activity per amount of total protein.

    Ex. A crude cell lysate might have a specific activity of 0.2 units/mg or ml protein upon which purification may increase to 10 units/mg or ml protein.

    One unit would be formation of one mol product per minute at a specific pH and temperature with a substrate concentration much greater than the value of Km.

  • Summary of Simple Saturation KineticsMichaelis-Menten ApproachBriggs-Haldane ApproachUse experimental data to obtain parameters of Michaelis-Menten kinetics if they are unknown.

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