lab training – enzyme kinetics ??09‐22 1 lab training – enzyme kinetics photometry qing...

Download Lab training – Enzyme Kinetics   ??09‐22 1 Lab training – Enzyme Kinetics  Photometry Qing Cheng Qing.Cheng@ki.se Biochemistry Division, MBB, KI Lab lecture Lab lecture - Enzyme Kinetics

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  • 20140922

    1

    Lab training Enzyme Kinetics & Photometry

    Qing Cheng

    Qing.Cheng@ki.seBiochemistry Division, MBB, KI

    Lab lecture

    Lab lecture - Enzyme Kinetics and Photometry

    Introduction on enzyme and kinetics Order of a reaction, first order kinetics Michaelis-Menten kinetics KM, Vmax and kcat Lineweaver-Burk plot Enzyme inhibition, competitive and non-competitive inhibition

    Spectrophotometer and Beer-Lambert Law

    Lab procedure Lab execution Lab report

    Safety in the lab

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    Lab lecture - Enzyme Kinetics and Photometry

    Introduction Enzyme

    G (withenzyme)

    G (withoutenzyme)

    Substrate

    Product

    Transitionstate,S

    Free

    ene

    rgy

    Reactionprogress

    Gforthereaction

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    Enzymes are biological catalysts characterized by Catalytic efficiency Specificity Regulated activity

    Enzyme-catalyzed reactions are affected by Enzyme concentration Substrate concentration Temperature pH Inhibitors Activators

    SubstrateEnzyme

    Product

    Lab lecture - Enzyme Kinetics and Photometry 20140922

    Kinetics is the study of chemical reaction rate (v, stands for velocity)

    Enzyme kinetics is the study of enzyme catalyzed reaction

    Determination of kinetics parameter measurement of enzyme activity

    First order kinetics v k S Zero order kinetics v k S

    Introduction Kinetics

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    Lab lecture - Enzyme Kinetics and Photometry

    Introduction Order of reactoin

    S k P

    Reactio

    nrate,v

    Substrateconcentration[S]

    Zeroorderreaction

    Firstorderreaction

    k

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    First order reaction Reaction rate is proportional to the substrates concentration. This is

    true when the substrate concentration is low during the reaction, and the substrate is the determine factor for the reaction rate.

    Zero order reaction The reaction rate is independent of the substrate concentration. This is

    true when the substrate concentration is much higher than the enzyme concentration during the reaction, and the enzyme is the determine factor for the reaction rate.

    Michaelis-Menten kinetics

    To understand how enzyme functions, we need a kinetic description of their activity.

    The reaction rate rises linearly as substrate concentration increases (first order reaction) and then begins to level off and approach a maximum at higher substrate concentration (zero order reaction)

    For many enzymes, the reaction rate V0 is defined as the number of moles of product formed per unit time when [P] is low, that is at times close to zero (hence, V0)

    Lab lecture - Enzyme Kinetics and Photometry

    Reactio

    nrate,v

    Substrateconcentration[S]

    Firstorderreaction

    Zeroorderreaction

    Reactio

    nrate,v

    Time

    [S1]

    [S2]

    [S3]

    [S4]V0

    V0

    V0V0

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    Michaelis-Menten kinetics

    Consider an enzyme that catalyzes the S to P by the following pathway:

    Lab lecture - Enzyme Kinetics and Photometry

    V0 = k2[ES] Rate of ES formation =k1[E][S] Rate of ES breakdown = (k2+k3)[ES]

    V0 is measured when [P] is low, therefore k4 becomes negligible.

    Steady state: When [ES] is formed and broken down at the same speed

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    Michaelis-Menten kinetics

    Simplify the previous equation by define a new constant, KM, called Michaelis constant

    KM has the units of concentration KM is independent of either [E] or [S]

    Lab lecture - Enzyme Kinetics and Photometry

    Solving [ES]

    (because at maximum rate, [ES]=[Etot])

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    Michaelis-Menten kinetics

    At very low substrate concentration ( ) : The reaction rate is directly proportional to the substrate concentration

    At very high substrate concentration ( ): The reaction rate is maximal, independent of substrate concentration.

    When KM is equal to the substrate concentration ( ): KM is equal to the substrate concentration at which the reaction rate is half

    its maxim value

    Lab lecture - Enzyme Kinetics and Photometry

    Reactionrate

    Substrateconcentration[S]

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    The lower the KM valueThe more efficient the enzyme

    Lineweaver-Burk plot

    Vmax is difficult to estimate because the initial reaction rate approaching Vmax asymptotically withincreasing substrate concentration. In addition, the high concentration of substrate often inhibitsreaction rate. To solve this problem, Lineweaver and Burk (1934) had inverted the Michaelis-Menten equation, which is referred as Lineweaver-Burk plot (or Double reciprocal plot):

    Lab lecture - Enzyme Kinetics and Photometry

    In this equation, 1/V and 1/[S] arevariables, while KM/Vmax and 1/Vmax areconstants. This can be plotted as alinear equation (y = ax + b).Specifically, 1/v is y, 1/[S] is x, KM/Vmaxis a (slope) and 1/Vmax is b (y-intercept). We can accurately calculateKM and Vmax value from a Lineweaver-Burk plot.

    y = ax + b

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    Nonlinear regression

    Indeed, KM and Vmax values can be calculateddirectly from the Michaelis-Menten equationthrough nonlinear regression.

    Lab lecture - Enzyme Kinetics and Photometry

    y = ax/(b+x)

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    http://www.colby.edu/chemistry/PChem/scripts/lsfitpl.html(in short: http://bit.ly/1re1XU4)

    Input the data pairs (V and [S])

    Choose fit function: ax/(b+x)

    Leave Parameter guesses as it is.

    Choose Convergence Mode: Damped or Strongly damped

    Click Fit or Fit & Plot (Java needed for plotting)

    Nonlinear regression

    Indeed, KM and Vmax values can be calculateddirectly from the Michaelis-Menten equationthrough nonlinear regression.

    Lab lecture - Enzyme Kinetics and Photometry

    y = ax/(b+x)

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    http://www.colby.edu/chemistry/PChem/scripts/lsfitpl.html(in short: http://bit.ly/1re1XU4)

    Input the data pairs (V and [S])

    Choose fit function: ax/(b+x)

    Leave Parameter guesses as it is.

    Choose Convergence Mode: Damped or Strongly damped

    Click Fit or Fit & Plot (Java needed for plotting)

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    Inhibition of enzyme activity

    Competitive inhibition

    Noncompetitive inhibition

    Lab lecture - Enzyme Kinetics and Photometry

    Enzyme Enzyme

    Competitiveinhibitor

    Enzyme

    Noncompetitiveinhibitor

    Substrate

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    Inhibition of enzyme activity Competitive inhibition

    Lab lecture - Enzyme Kinetics and Photometry

    Enzyme

    Competitiveinhibitor

    Noinhibitor

    CompetitiveinhibitorNoinhibitor

    +Competitiveinhibitor

    Vmax isnotaffectedKM isincreased

    +I

    EI

    E + S ES E + P

    ki SI

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    Inhibition of enzyme activity Noncompetitive inhibition

    Lab lecture - Enzyme Kinetics and Photometry

    Noinhibitor

    Noncompetitiveinhibitor

    S IE + I ES E + Pki SEI EIS

    Noinhibitor

    +Noncompetitiveinhibitor

    Enzyme

    Noncompetitiveinhibitor

    Vmax isdecreasedKM isnotaffected

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    Inhibition of enzyme activity

    Lab lecture - Enzyme Kinetics and Photometry

    Noinhibitor

    Mixinhibition

    Vmax isdecreasedKM isnotaffected

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    Noinhibitor

    Competitiveinhibitor

    Noinhibitor

    Noncompetitiveinhibitor

    Vmax isnotaffectedKM isincreased

    Vmax isdecreasedKM isincreased

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    Lab lecture - Enzyme Kinetics and Photometry

    Photometry - Spectrophotometer

    How to measure the chemical reactions rate Different molecules have different

    absorption Some molecules (e.g. proteins) have

    several absorbance peaks during the wave scan

    Spectrophotometer

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    LightsourceFilterSampleDetectorReadout

    Lab lecture - Enzyme Kinetics and Photometry

    Photometry - Beer-Lambert law

    Beer-Lambert law is a mathematical means of expressing how light is absorbed bymatter. The law states that the amount of light emerging from a sample isdiminished by three physical phenomena:

    The concentration of absorbing sample in its pathway (C, in unit of molarity, M) The distance the light travels through the sample (, in units of centimeters, cm) The probability that the light of that particular wavelength will be absorbed by

    the material, also known as molar absorption (or extinction) coefficient (), inunits that are reciprocals of molarity and distance in centimeters, M-1cm-1)

    T: 0 - 1A: - 0

    Due to technical limitation, the best readingrange of spectrophotometer is normallyfrom 0.1 1, thus:

    o If A is too high, dilute the sampleo If A is too low, concentrate the sample

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    Lab lecture - Enzyme Kinetics and Photometry

    Lab training

    Enzyme: Alkaline Phosphatase (ALP) Remove phosphate groups from many types of molecules. Function as a dimer, and take effect under alkaline conditions Made in liver, bone, and other tissues. It can be measured in a routine blood test. Abnormally high serum

    levels of ALP may indicate bone, liver disease, etc.

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    Lab lecture - Enzyme Kinetics and Photometry

    Lab training Outline

    Determine the following parameters of alkaline phosphataseusing p-nitro-phenyl-phosphate (NPP) as substrate Optimal pH KM Vmax Inhibition

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    p-Nitro-Phenyl-Phosphate (NPP)

    p-Nitrophenol

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    Lab lectur