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

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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

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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

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