03 practical enzymology - freelascu.free.fr/enzymology/03 practical enzymology.pdf ·...

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

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.

• 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!

CONTINUS AND DISCONTINUS ASSAYS

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 5 10 15 20

S, mMS2 (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 ]

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!)

ATPdépôt

Radioactivity measurement

[γ-32P]-ATP � ADP + 32Pi secondary reaction enzymatic(contaminanting phosphatase)

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)

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….)

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

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

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

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!)

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 »)

Chemiluminescence

Light emission

A + B � C + hνHigh sensitivity (similar to Radioactivity)

Not an absolute method (callibration needed)

Immunochemical measurements (ELISA, western blot)

Bio-chemiluminescence

THE GLOW-WORM

Polarography: following the glucose oxidase reaction with the Clarck electrode

O2H2O2

FAD FADH2

Glucoseoxidase

This method is used for measuring the glycemy (blood sugar)

2 problems:

Polarographie:Mesure de la vitesse d’oxydation du glucose catalysée par laGlucose oxydase: électrode de Clark

OHCOO-

FAD FADH2Glucoseoxydase

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

Polarographie:Mesure de la vitesse d’oxydation du glucose catalysée par laGlucose oxydase: électrode de Clark

OHCOO-

FAD FADH2Glucoseoxydase

The enzyme is specific for β-D-glucose but equilibrium is fast

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!)

OPO42-

hexokinase

glucose-6-phosphate dehydrogenase

6-phosphogluconolactone

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 » ?

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.

The pyruvate kinase reaction

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

The kinase (ATPase) 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

O2H2O2

FAD FADH2

Glucoseoxydase

Substratereduced

Productoxydized

PeroxydaseTo be added in excess

O2H2O2

Substratereduced

Productoxydized

3,3'-Diaminobenzidine

Full chemical name: 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)

Oxidasereaction

O2H2O2

Substratereduced

Productoxydized

Oxidasereaction

4-chlorophenol and 4-aminophenazone (4-AA)

Any reaction generating ATP can be followed by coupling with the peroxydase reaction

Chromogenic substrates

β-galactosidase

Artificial substrates: on of the products is coloured or fluorescent

THROMBINE

Phosphatases:

Protéases:

SO2 Gly-Pro-Arg NH NO2

THROMBINE

SO2 Gly-Pro-Arg NH NO2

SO2 Gly-Pro-Arg-COO- NH2 NO2+

Fibrinogen is the physiological substrate

Fluorogenic fluorogènes

Phosphatases:

6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)

Umbelliferone or 7-hydroxycoumarine

Chemical reaction of one product

Exemple –cholineaterase reaction

CH2CH2 S C

CH3NCH3

CH2CH2 SH

CH3 COO-

CH2CH2 SH

-OOC S COO-

SH COO-

S COO-

reaction with dithio-bis-nitrobenzoate

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

• 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.

= KMVmax

+ 1Vmax

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.

= KMVmax

+ 1Vmax

0 20 40 60 80 100

Data 1

y = m1*m0/(m2+m0)

ErrorValue2.8222227.06Vmax

0.8641821.318Km

NA123.44ChisqNA0.99913R

0 20 40 60 80 100

Data 1

y = m1*m0/(m2+m0)

0.8641821.318Km

NA123.44ChisqNA0.99913R

0 20 40 60 80 100

Data 1

residual0

Data 1

sum y = m1*m0/(m2+m0)

0.8641821.318KmNA123.44ChisqNA0.99913R

0 20 40 60 80 100

50, 5 200, 40

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

Practical enzymology

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

Direct Assay• Direct measurement of [P] or [S] as a function of time

• eg. cytochromec oxidase

cyt c (Fe2+) cyt c (Fe3+)

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

O CO2-

dihydroorate(reduced)

orotic acid(oxidized)

dihydroorate dehydrogenase

ubiquinone(oxidized)

ubiquinol(reduced)

2,6-dichlorophenol indophenol(dye reagent)

oxidized (dark blue)reduced(colourless)

Coupled Assay• Enzymatic reaction of interest is paired with a 2nd enzymatic

reaction which may be easily followed.

• eg. Hexokinase

• Caveats:

OPO42-

hexokinase

glucose-6-phosphate dehydrogenase

6-phosphogluconolactone

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

Detection Methods• Spectrophotometry

Abs = ε × l × c (Beer’s law)vi = dc/dt = dAbs/dt (1/(ε × l))

• Spectrofluorimetry

• Radioactivity

03 practical enzymology - freelascu.free.fr/enzymology/03 practical enzymology.pdf ·...

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