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1
PRACTICAL ENZYMOLOGY
S � P
A1. Decrease of S or increase of P: How can be the reaction followed ?
A2. Calculations: how can v be obtained from experimental data?
v generally in µmol/min/ml (= UI/ml) NOT µM!!!
kcat in s-1
Measurement of reaction RATE
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Specificity of the reaction measurement (“do we measure the correct reaction”?)
Is the reaction you are measuring carried out by only one enzyme?
Temperature? Co-factors? Competing activities?
Are there “non-enzymatic” pathways to the products?
Controls, controls, controls.
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• Continuous assay: The signal is measured at discrete intervals over the entire linear range of the reaction. The initial velocity is measured from the slope of the linear range of the curve.
• Discontinuous (End-point) assay: the signal is measured at a specific time point on the linear range of the assay.
Disadvantage : won’t notice deviations from linearity!
time
time
CONTINUS AND DISCONTINUS ASSAYS
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PRACTICAL ENZYMOLOGY
S � P
The reaction rate SHOULD be proportionnal to the enzyme concentration
If the reaction progress is not linear in function of time, one should measure the tangent at zero time
0
0,01
0,02
0,03
0,04
0,05
0,06
0 5 10 15 20
S, mMS2 (mM)
S (
mM
)
Temps (min)
Why the reaction is not linear?
Decrease of [ s ],orAccumulation of P a.reverse reactionb.product inhibition
Vmax = kcat * [ E ] t; or v = kcat* [ES ]
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PRACTICAL ENZYMOLOGYS � P
a. With separation (no change in a parameter needed)
Glucose + [γ-32P]-ATP � [32P]-G-6-P+ ADPMg2+
Stop at various times (EDTA or denaturation by acid or heat)Separation by TLC (thin layer chromatography or HPLC)(Ion exchange; DEAE Cellulose)
<10% of the substrate should react (initial rate!)
Pi
G-6-P
ATPdépôt
Radioactivity measurement
[γ-32P]-ATP � ADP + 32Pi secondary reaction enzymatic(contaminanting phosphatase)
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PRACTICAL ENZYMOLOGYS � P
[γ-32P]-ATP + GDP � [γ-32P]-GTP + ADP
Site-directed Mutation of Nucleoside diphosphate kinase
Wrong analysis – not initial rates!
ADP and GDP are not seen (are not radioactive)
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PRACTICAL ENZYMOLOGY
S � P
b. Continuous assays, no separation (a parameter change during the reaction)
This parameter should be proportionnal to the concentration
• Absorbance• fluorescence intensity• Chemiluminescence• Oxygen concentration• pH• (RMN, RPE, viscosimetry, calorimetry….)
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PRACTICAL ENZYMOLOGY
b. Continuous assays, no separation
Example: dehydrogenases NAD(P)-dependentes
NADH and NADPH absorb lightat 340 nm and are fluorescent
NAD+ and NADP+ do NOT absorb lightat 340 nm and are NOT fluorescent
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PRACTICAL ENZYMOLOGY
b. Continuous assays, no separation
Example: dehydrogenases NAD(P)-dependentes
Spectrophotometric assay (decrease absorbance at 340 nm)
Abs = ε × l × c (Beer’s law)∆Abs/time = ε × l × ∆c/time∆c mM = mmol/l = µmol/mlRate v = µmol/min/ml or IU/mlSpecific activity: IU/mg
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PRACTICAL ENZYMOLOGY
Spectrophotometric assay (decrease absorbance at 340 nm)
Abs = ε × l × c (Beer’s law)∆Abs/time = ε × l × ∆c/time∆c µmol/mlRate v = µmol/min/ml or IU/ml
Turnover calculus: Vmax = kcat * [ E ]tLDH specific activity about 400 IU/mgThe enzyme is a tetramer, each subunit Mr 350001 mg: 0.001g/35000 = 28.5x10-9 mol = 0.0285 µmol300 IU/mg = (300 µmol/min)/(0.0285 µmol) 10500 min-1/60 = 175s-1
THE TURNOVER NUMBER SHOULD BE CALCULATED PER SUBUNIT
12http://www.strenda.org/documents.html
Assay ConditionsMeasured reaction As a stoichiometrically balanced equation.Assay temperature Assay pHBuffer & concentrations e.g., 100 mM Tris-HCI, 200 mM potassiumMetal salt(s) & concentrations e.g., 10 mM KCI, 1.0 mM MgS04Other assay components e.g., 10 mM EDTA, 1.0 mM dithiothreitolSubstrates & concentrations e.g., 100 mM glucose, 5 mM ATP (coupled assay components)Enzyme/protein concentration e.g., nmol/ml or mg/ml
ActivityInitial rates of the reaction measured Proportionality between initial velocity and enzyme concentration Specific activity
Units necessary:Expressed as concentration per amount per time, e.g. micromol product formed/(min . mg enzyme protein) -sometimes referred to as enzyme unit or international unit). The katal (mol/second) may alternatively be used as a unit of activity (conversion factor 1 unit = 16.67 nkat).
Much reported enzyme data is of limited use to others attempting to apply those data, because the conditions under insufficiently documented. This list was compiled, as a service to the community, by the STRENDA Commission to c information that should accompany any published enzyme activity data
Standard Requirements for Reporting Enzyme Activity Data
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Measuring fluorescence intensity
More sensitive than spectrophotometryBut…Is not an absolute method (calibration is needed)
G-6-P + NADP+ + H+ � Phosphogluconic acid + NADPH
The reverse reaction cannont be easily followed(Inner filter effect if A340 > 0.1) Light absorbtion cannot be avoided(no absorbtion, no fluorescence!)
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Measuring fluorescence intensity
The reverse reaction cannont be easily followed(Inner filter effect if A340 > 0.1)
excitation
emission
Light absorbtion cannot be avoided(no absorbtion, no fluorescence!)
2 solutions:Correction by calculusA different fluorimeter geometry (« face-front »)
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Chemiluminescence
Light emission
A + B � C + hνHigh sensitivity (similar to Radioactivity)
Not an absolute method (callibration needed)
Immunochemical measurements (ELISA, western blot)
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Bio-chemiluminescence
THE GLOW-WORM
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Polarography: following the glucose oxidase reaction with the Clarck electrode
OOH
CH2OH
OH
OH
OH
O
O
CH2OH
OH
OH
OH
OH
COO-
CH2OH
OH
OH
OH
O2H2O2
FAD FADH2
Glucoseoxidase
This method is used for measuring the glycemy (blood sugar)
2 problems:
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Polarographie:Mesure de la vitesse d’oxydation du glucose catalysée par laGlucose oxydase: électrode de Clark
OOH
CH2OH
OH
OH
OH
OO
CH2OH
OH
OH
OH
OHCOO-
CH2OH
OH
OH
OH
O2H
2O
2
FAD FADH2Glucoseoxydase
This method is used for measuring the glycemy (blood sugar)
1. GOD has a very high Km value; one cannot measure the end point! [S]<<Km, so generally the initial rate is measured, which is proportionnal with the substrate concentrationv = (Vmax/Km) S
2. The enzyme is specific for β-D-glucose
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Polarographie:Mesure de la vitesse d’oxydation du glucose catalysée par laGlucose oxydase: électrode de Clark
OOH
CH2OH
OH
OH
OH
OO
CH2OH
OH
OH
OH
OHCOO-
CH2OH
OH
OH
OH
O2H
2O
2
FAD FADH2Glucoseoxydase
This method is used for measuring the glycemy (blood sugar)
The enzyme is specific for β-D-glucose but equilibrium is fast
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Measuring protons released/bound during the reaction
Glucose + MgATP � G-6-P+ ADP + H+
Measuring H+ release:
1.Manometric (oldest method): reaction with NaHCO3 and measuring pressure. Glycolysis and Krebs cycle were discovered in that way in the 1930. No spectrophotometer, no X-ray at that time!!2.Measuring pH in a buffer-free medium3.pH-stat (NaOH is added to maintain pH constant)4.Using a pH indicator dye
Otto von WARBURGKaiser-Wilhelm-Institut (now Max-Planck-Institut) für BiologieBerlin-Dahlem, Germany1883-1970http://nobelprize.org/nobel_prizes/medicine/laureates/1931/warburg-bio.html
No absorbance or other signal needed (=no possible artifact!)
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+ ATP
O
H
HO
H
HO
H
OH
OHHH
OH
O
H
HO
H
HO
H
OH
OHHH
OPO42-
+ ADP
hexokinase
glucose-6-phosphate dehydrogenase
6-phosphogluconolactone
NADP+
NADPH
Continuous assays even if no parameter can be measured: coupled reactions
Absorbance or fluorescence intensity measurement
Glucose-6-phosphate dehydrogenase added in large excess
What means: « added in large excess » ?
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Continuous assays even if no parameter can be measured: coupled reactions
What means: « added in large excess » ?The measured rate should be that of the hexokinase reaction!The G6PDH activity should be >100x that of the HKBut…..in fact the important parameter is Vmax/Km and not Vmax since [G6P] << Km!!!
How to be sure that the coupling enzyme is in excess? You add more G6PDH and the measured reaction rate do not change.
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The pyruvate kinase reaction
Continuous assays even if no parameter can be measured: coupled reactions
pyruvate kinase
Mg2+, K+
Pyruvate + NADH + H+ � Lactate + NAD+
The pyruvate formed in the PK reaction is detected with the LDH added in large excess
The decrease absorbance is measured
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The kinase (ATPase) reactions
Continuous assays even if no parameter can be measured: coupled reactions
ADP + PEP � ATP + Pyruvate
Pyruvate + NADH + H+ � Lactate + NAD+
The ADP formed in the reaction is detected with the PK and LDH reactions; ATP is regenerated
The decrease absorbance is measured
ATP + X � ADP + X-P
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Continuous assays even if no parameter can be measured: coupled reactions
OOH
CH2OH
OH
OH
OH
O
O
CH2OH
OH
OH
OH
OH
COO-
CH2OH
OH
OH
OH
O2H2O2
FAD FADH2
Glucoseoxydase
Substratereduced
Productoxydized
H2O
PeroxydaseTo be added in excess
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Continuous assays even if no parameter can be measured: coupled reactions
O2H2O2
Substratereduced
Productoxydized
H2O
PeroxydaseTo be added in excess
3,3'-Diaminobenzidine
ABTS
Full chemical name: 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)
Oxidasereaction
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Continuous assays even if no parameter can be measured: coupled reactions
O2H2O2
Substratereduced
Productoxydized
H2O
PeroxydaseTo be added in excess
Oxidasereaction
4-chlorophenol and 4-aminophenazone (4-AA)
Any reaction generating ATP can be followed by coupling with the peroxydase reaction
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Chromogenic substrates
β-galactosidase
β-galactosidase
+
+
Artificial substrates: on of the products is coloured or fluorescent
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Chromogenic substrates
THROMBINE
Phosphatases:
Protéases:
SO2 Gly-Pro-Arg NH NO2
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Chromogenic substrates
THROMBINE
SO2 Gly-Pro-Arg NH NO2
SO2 Gly-Pro-Arg-COO- NH2 NO2+
Fibrinogen is the physiological substrate
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Fluorogenic fluorogènes
Phosphatases:
6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)
Umbelliferone or 7-hydroxycoumarine
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Chemical reaction of one product
Exemple –cholineaterase reaction
NCH3
CH3
CH3
CH2CH2 S C
O
CH3NCH3
CH3
CH3
CH2CH2 SH
CH3 COO-
NCH3
CH3
CH3
CH2CH2 SH
S
O2N
-OOC S COO-
NO2
SH COO-
NO2
S COO-
NO2
SR
reaction with dithio-bis-nitrobenzoate
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The enzymologist should find under which experimental conditions the equations are most simple, rather than develop huge equations for data obtained under non optimised conditions
Analysis of Kinetic Data
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Analysis of Kinetic Data
• The rate equations are non-linear, so it is convenient to reformulate the equation to give a linear relationship. The most common linearization is the Lineweaver-Burk or double reciprocal plot, obtained from the reciprocal of the Michaelis-Menten equation.
• DISADVANTAGE: the data usually involve large ratios of KM so the data is crowded. At low [S] values the errors are often large.
1ν0
= KMVmax
1[S]
+ 1Vmax
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Analysis of Kinetic Data
• The rate equations are non-linear, so it is convenient to reformulate the equation to give a linear relationship. The most common linearization is the Lineweaver-Burk or double reciprocal plot, obtained from the reciprocal of the Michaelis-Menten equation.
• DISADVANTAGE: the data usually involve large ratios of KM so the data is crowded. At low [S] values the errors are often large.
1ν0
= KMVmax
1[S]
+ 1Vmax
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0
50
100
150
200
0 20 40 60 80 100
Data 1
sum
sum
[S]
y = m1*m0/(m2+m0)
ErrorValue2.8222227.06Vmax
0.8641821.318Km
NA123.44ChisqNA0.99913R
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0
50
100
150
200
0 20 40 60 80 100
Data 1
sum
sum
[S]
y = m1*m0/(m2+m0)
ErrorValue2.8222227.06Vmax
0.8641821.318Km
NA123.44ChisqNA0.99913R
-4
-2
0
2
4
6
8
0 20 40 60 80 100
Data 1
residual0
resi
dual
[S]
40
0
50
100
150
200
Data 1
sum
sum y = m1*m0/(m2+m0)
ErrorValue2.8222227.06Vmax
0.8641821.318KmNA123.44ChisqNA0.99913R
-4
-2
0
2
4
6
8
0 20 40 60 80 100
resi
dual
s
[S]
50, 5 200, 40
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En résumé : quelques définitions : - le Becquerel (Bq) : Unité de radioactivité du Système International. - la désintégration par seconde (dps) :
1 Bq = 1 dps - la désintégration par minute (dpm) :
1 dps correspond à 60 dpm - le Curie (Ci) : nombre de dps pour 1 g de radium
1 Ci = 3,7 1010 Bq = 2,22.10 12 dpm - le nombre de coups par minute (cpm) : mesure de la radioactivité effectuée par un compteur de radioactivité. NB : Il n'y a pas équivalence absolue entre le nombre de cpm et de dpm. Pour connaître l'équivalence entre cpm et dpm, il faut connaître le rendement du compteur. Le rendement de comptage sera le rapport cpm/dpm (ce rendement peut être > à 1)
rendement = cpm / dpm
dpm = cpm / rendement - La période (t 1/2) d'un radioélément est le temps pour lequel la moitié des atomes initiaux disparaissent. Isotopes les plus intéressants :
Isotope : 3H 14C 32P
Rayonnement : β€ β€ β€
Emax (MeV) : 0,0186 0,156 1,71
t1/2 : 12,4 ans 5730 ans 14,3 jours
43Fersht table 6.1 t1/2
Isotope t1/2 Ci/mol Type ofemission
Max energy MeV
14C 5730 yr 62.4 ββββ 0.156
3H 12.35 yr 29 000 ββββ 0.0186
35S 87.4 d 1 490 000 ββββ 0.167
32P 14.3 d 9 130 000 ββββ 1.7
125I 60 d 2 180 000 γγγγ
131I 8.06 d 16 200 000 ββββ, γγγγ
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Practical enzymology
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Practical Enzyme Kinetics
• There are many ways to assay an enzyme!
• Assays differ in their features and in their uses and limitations. It is important to understand the terminologies used in describing an enzyme assay and to keep the limitations in mind when you read papers reporting values obtained using enzyme kinetics or when you seek to assay an enzyme for yourself.
• The following methods are described for assays designed to measure initial velocities. Recall that working under initial velocity conditions greatly
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Direct Assay• Direct measurement of [P] or [S] as a function of time
• eg. cytochromec oxidase
cyt c (Fe2+) cyt c (Fe3+)
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Indirect Assay
• In this case, S & P do not provide a distinct, measureablesignal. Product formation is, therefore, monitored by coupling the reaction to a non-enzymatic reaction that doesyield a distinct signal.
• eg. dihydroorate dehydrogenase
HN
NH
O
O CO2-
H
H
H HN
NH
O
O CO2-
H
dihydroorate(reduced)
orotic acid(oxidized)
dihydroorate dehydrogenase
CH3
R
OH
H3CO
H3CO
O
CH3
R
OH
H3CO
H3CO
OH
ubiquinone(oxidized)
ubiquinol(reduced)
N
N
O
Cl
Cl
NH
N
OH
Cl
Cl
2,6-dichlorophenol indophenol(dye reagent)
oxidized (dark blue)reduced(colourless)
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Coupled Assay• Enzymatic reaction of interest is paired with a 2nd enzymatic
reaction which may be easily followed.
• eg. Hexokinase
• Caveats:
+ ATP
O
H
HO
H
HO
H
OH
OHHH
OH
O
H
HO
H
HO
H
OH
OHHH
OPO42-
+ ADP
hexokinase
glucose-6-phosphate dehydrogenase
6-phosphogluconolactone
NADP+
NADPH
NAD+
NADH
3
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Continuous vs. End-point assays• Continuous assay: The
signal is measured at discrete intervals over the entire linear range of the reaction. The initial velocity is measured from the slope of the linear range of the curve.
• Discontinuous (End-point) assay: the signal is
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Detection Methods• Spectrophotometry
Abs = ε × l × c (Beer’s law)vi = dc/dt = dAbs/dt (1/(ε × l))
• Spectrofluorimetry
• Radioactivity