PRACTICAL ENZYMOLOGY Measurement of reaction RATE 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 kcat
in µmol/min/ml (= UI/ml) NOT µM!!! in s-1 1
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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CONTINUS AND DISCONTINUS 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. time
• 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
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PRACTICAL ENZYMOLOGY S
P
Vmax = kcat * [ E ]t; or v = kcat* [ES ]
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 0,06
S, mM S2 (mM)
0,05
Why the reaction is not linear? Decrease of [ s ], or Accumulation of P a.reverse reaction b.product inhibition
S (mM)
0,04 0,03 0,02 0,01 0 0
5
10
Temps (min)
15
20
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PRACTICAL ENZYMOLOGY S P a. With separation (no change in a parameter needed) Mg2+
Glucose + [γ-32P]-ATP
[32P]-G-6-P+ ADP
Stop at various times (EDTA or denaturation by acid or heat) Separation by TLC (thin layer chromatography or HPLC) (Ion exchange; DEAE Cellulose)
[γ-32P]-ATP ADP + 32Pi secondary reaction enzymatic (contaminanting phosphatase) Pi G-6-P
Radioactivity measurement
ATP dépôt
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)
Light absorbtion cannot be avoided (no absorbtion, no fluorescence!)
excitation
emission
2 solutions: Correction by calculus A different fluorimeter geometry (« face-front ») 14
Chemiluminescence A+B
C + hν
High sensitivity (similar to Radioactivity) Light emission
Not an absolute method (callibration needed)
Immunochemical measurements (ELISA, western blot) 15
Bio-chemiluminescence
THE GLOW-WORM 16
Polarography: following the glucose oxidase reaction with the Clarck electrode
CH 2OH
CH 2OH O OH
OH OH
CH 2OH O
Glucose oxidase
OH OH
OH
OH O
OH OH
OH
FAD
H 2O 2
COO-
OH
FADH2
O2
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 la Glucose oxydase: électrode de Clark This method is used for measuring the glycemy (blood sugar)
CH2OH O OH
CH2OH O
OH
OH
OH
OH
OH
FAD
HO 2 2
CH2OH OH OH COO-
O
OH
FADH 2
O
OH OH
Glucose oxydase
2
1. GOD has a very high Km value; one cannot measure the end point! [S] à 1) rendement = cpm / dpm
dpm = cpm / rendement
- La période (t1/2) d'un radioélément est le temps pour lequel la moitié des atomes initiaux disparaissent.
Isotopes les plus intéressants : Isotope : 3
H C 32 P 14
Rayonnement :
Emax (MeV) :
β β β
0,0186 0,156 1,71
t1/2 : 12,4 ans 5730 ans 14,3 jours
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Ci/mol
Type of emission
Max energy MeV
Isotope
t1/2
14C
5730 yr
62.4
β
0.156
3H
12.35 yr
29 000
β
0.0186
35S
87.4 d
1 490 000
β
0.167
32P
14.3 d
9 130 000
β
1.7
125I
60 d
2 180 000
γ
131I
8.06 d
16 200 000
β, γ
Fersht table 6.1 t1/2
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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 45 working under initial velocity conditions greatly
Direct Assay • Direct measurement of [P] or [S] as a function of time • eg. cytochrome c oxidase cyt c (Fe2+)
cyt c (Fe3+)
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Indirect Assay dihydroorate (reduced) O
• In this case, S & P do not provide a distinct, measureable signal. Product formation is, therefore, monitored by coupling the reaction to a nonenzymatic reaction that does yield a distinct signal. • eg. dihydroorate dehydrogenase
orotic acid (oxidized) O
H
H O
N H
H
dihydroorate dehydrogenase
H
HN
HN O
CO2-
N H
O
CO2-
OH CH3
H3CO
H3CO
CH3
H3CO
R
H3CO
R
OH
OH
ubiquinone (oxidized)
ubiquinol (reduced)
N N
NH
N Cl
Cl
reduced (colourless)
oxidized (dark blue) OH Cl
O Cl
2,6-dichlorophenol indophenol (dye reagent)
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Coupled Assay • Enzymatic reaction of interest is paired with a 2nd enzymatic reaction which may be easily followed. H OH
• eg. Hexokinase
H O
H H
NADH
H
+ ADP H O
hexokinase
HO HO
NAD+
2H OPO4 3
+ ATP
HO HO H
OH OH
H
H OH OH
NADP+
glucose-6-phosphate dehydrogenase
NADPH 6-phosphogluconolactone
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• Caveats:
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
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