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PRACTICAL ENZYMOLOGY

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 in µmol/min/ml (= UI/ml) NOT µM!!! k cat in s -1.

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PRACTICAL ENZYMOLOGY

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  1. 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 in µmol/min/ml (= UI/ml) NOT µM!!! kcat in s-1

  2. 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.

  3. 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. • 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!

  4. 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 Why the reaction is not linear? Decrease of [ s ], or Accumulation of P a.reverse reaction b.product inhibition

  5. PRACTICAL ENZYMOLOGY Mg2+ Glucose + [g-32P]-ATP  [32P]-G-6-P+ ADP Pi Radioactivity measurement G-6-P ATP dépôt S  P • With separation (no change in a parameter needed) Stop at various times (EDTA or denaturation by acid or heat) Separation by TLC (thin layer chromatography or HPLC) (Ion exchange; DEAE Cellulose) [g-32P]-ATP  ADP + 32Pi secondary reaction enzymatic (contaminanting phosphatase) <10% of the substrate should react (initial rate!)

  6. PRACTICAL ENZYMOLOGY S  P ADP and GDP are not seen (are not radioactive) [g-32P]-ATP + GDP  [g-32P]-GTP + ADP Site-directed Mutation of Nucleoside diphosphate kinase Wrong analysis – not initial rates!

  7. 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….)

  8. PRACTICAL ENZYMOLOGY b. Continuous assays, no separation Example: dehydrogenases NAD(P)-dependentes NADH and NADPH absorb light at 340 nm and are fluorescent NAD+ and NADP+ do NOT absorb light at 340 nm and are NOT fluorescent

  9. PRACTICAL ENZYMOLOGY b. Continuous assays, no separation Example: dehydrogenases NAD(P)-dependentes Spectrophotometric assay (decrease absorbance at 340 nm) Abs = e l  c (Beer’s law) DAbs/time = e l Dc/time Dc mM = mmol/l = µmol/ml Rate v = µmol/min/ml or IU/ml Specific activity: IU/mg

  10. PRACTICAL ENZYMOLOGY Spectrophotometric assay (decrease absorbance at 340 nm) Abs = e l  c (Beer’s law) DAbs/time = e l Dc/time Dc µmol/ml Rate v = µmol/min/ml or IU/ml Turnover calculus: Vmax = kcat * [ E ]t LDH specific activity about 400 IU/mg The enzyme is a tetramer, each subunit Mr 35000 1 mg: 0.001g/35000 = 28.5x10-9 mol = 0.0285 µmol 300 IU/mg = (300 µmol/min)/(0.0285 µmol) 10500 min-1/60 = 175s-1 THE TURNOVER NUMBER SHOULD BE CALCULATED PER SUBUNIT

  11. Standard Requirements for Reporting Enzyme Activity Data 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 Assay Conditions Measured reaction As a stoichiometrically balanced equation. Assay temperature Assay pH Buffer& concentrations e.g., 100 mM Tris-HCI, 200 mM potassium Metal salt(s) & concentrations e.g., 10 mM KCI, 1.0 mM MgS04 Other assay components e.g., 10 mM EDTA, 1.0 mM dithiothreitol Substrates & concentrations e.g., 100 mM glucose, 5 mM ATP (coupled assay components) Enzyme/protein concentration e.g., nmol/ml or mg/ml Activity Initial 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). http://www.strenda.org/documents.html

  12. Measuring fluorescence intensity More sensitive than spectrophotometry But… 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!)

  13. Measuring fluorescence intensity excitation emission The reverse reaction cannont be easily followed (Inner filter effect if A340 > 0.1) Light absorbtion cannot be avoided (no absorbtion, no fluorescence!) 2 solutions: Correction by calculus A different fluorimeter geometry (« face-front »)

  14. Chemiluminescence A + B  C + hn High sensitivity (similar to Radioactivity) Not an absolute method (callibration needed) Light emission Immunochemical measurements (ELISA, western blot)

  15. Bio-chemiluminescence THE GLOW-WORM

  16. Polarography: following the glucose oxidase reaction with the Clarck electrode C H O H 2 C H O H C H O H 2 2 O O O H O H O H O O H O H C O O - O H O H O H O H O H O H FAD FADH 2 O H O 2 2 2 Glucose oxidase This method is used for measuring the glycemy (blood sugar) 2 problems:

  17. Polarographie:Mesure de la vitesse d’oxydation du glucose catalysée par la Glucose oxydase: électrode de Clark C H O H 2 C H O H C H O H 2 2 O O O H O H O H O O H O H C O O - O H O H O H O H O H O H FAD FADH 2 O H O 2 2 2 This method is used for measuring the glycemy (blood sugar) Glucose oxydase 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 concentration v = (Vmax/Km) S 2. The enzyme is specific for b-D-glucose

  18. Polarographie:Mesure de la vitesse d’oxydation du glucose catalysée par la Glucose oxydase: électrode de Clark C H O H 2 C H O H C H O H 2 2 O O O H O H O H O O H O H C O O - O H O H O H O H O H O H FAD FADH 2 O H O 2 2 2 This method is used for measuring the glycemy (blood sugar) Glucose oxydase The enzyme is specific for b-D-glucose but equilibrium is fast

  19. 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 medium 3.pH-stat (NaOH is added to maintain pH constant) 4.Using a pH indicator dye No absorbance or other signal needed (=no possible artifact!) Otto von WARBURG Kaiser-Wilhelm-Institut (now Max-Planck-Institut) für Biologie Berlin-Dahlem, Germany 1883-1970 http://nobelprize.org/nobel_prizes/medicine/laureates/1931/warburg-bio.html

  20. Continuous assays even if no parameter can be measured: coupled reactions Glucose-6-phosphate dehydrogenase added in large excess Absorbance or fluorescence intensity measurement What means: « added in large excess » ?

  21. 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 HK But…..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.

  22. Continuous assays even if no parameter can be measured: coupled reactions pyruvate kinase Mg2+, K+ The pyruvate kinase reaction The pyruvate formed in the PK reaction is detected with the LDH added in large excess Pyruvate + NADH + H+ Lactate + NAD+ The decrease absorbance is measured

  23. Continuous assays even if no parameter can be measured: coupled reactions The kinase (ATPase) reactions ATP + X  ADP + X-P The ADP formed in the reaction is detected with the PK and LDH reactions; ATP is regenerated ADP + PEP  ATP + Pyruvate Pyruvate + NADH + H+ Lactate + NAD+ The decrease absorbance is measured

  24. Continuous assays even if no parameter can be measured: coupled reactions C H O H 2 C H O H C H O H 2 2 O O O H O H O H O O H O H C O O - Glucose oxydase O H O H O H O H O H O H FAD FADH 2 O H O 2 2 2 Peroxydase To be added in excess Product oxydized Substrate reduced H O 2

  25. Continuous assays even if no parameter can be measured: coupled reactions Oxidase reaction 3,3'-Diaminobenzidine O H O 2 2 2 Peroxydase To be added in excess Product oxydized Substrate reduced ABTS Full chemical name: 2,2'-azino-bis-(3- ethylbenzothiazoline-6-sulfonic acid) H O 2

  26. Continuous assays even if no parameter can be measured: coupled reactions Oxidase reaction O H O 2 2 2 Peroxydase To be added in excess Product oxydized Substrate reduced H O 2 Any reaction generating ATP can be followed by coupling with the peroxydase reaction 4-chlorophenol and 4-aminophenazone (4-AA)

  27. Chromogenic substrates Artificial substrates: on of the products is coloured or fluorescent b-galactosidase + + b-galactosidase

  28. Chromogenic substrates Phosphatases: Protéases: THROMBINE Gly-Pro-Arg

  29. Chromogenic substrates THROMBINE Fibrinogen is the physiological substrate Gly-Pro-Arg + Gly-Pro-Arg-COO-

  30. Fluorogenic fluorogènes Phosphatases: Umbelliferone or 7-hydroxycoumarine 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)

  31. Chemical reaction of one product Exemple –cholineaterase reaction reaction with dithio-bis-nitrobenzoate

  32. Analysis of Kinetic Data 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

  33. 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. • There are several other linear forms of the Michaelis-Menten equation. However, most data is now easily analyzed directly on computer by a direct non-linear regression fit to the Michaelis-Menten equation.

  34. 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. • There are several other linear forms of the Michaelis-Menten equation. However, most data is now easily analyzed directly on computer by a direct non-linear regression fit to the Michaelis-Menten equation.

  35. 50, 5 200, 40

  36. Isotope t1/2 Ci/mol Type of emission Max energy MeV 14C 5730 yr            62.4 b    0.156 3H 12.35 yr      29 000 b    0.0186 35S 87.4 d 1 490 000 b    0.167 32P 14.3 d 9 130 000 b    1.7 125I 60 d 2 180 000 g 131I 8.06 d 16 200 000 b, g             Fersht table 6.1 t1/2

  37. Practical enzymology

  38. 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 simplifies the interpretation of the kinetic data. The most satisfactory method of determining kcat, kcat/Km and Km is to measure initial rates (≤ 5% of the reaction).

  39. Direct Assay • Direct measurement of [P] or [S] as a function of time • eg. cytochrome c oxidase cyt c (Fe2+) cyt c (Fe3+)

  40. Indirect Assay • In this case, S & P do not provide a distinct, measureable signal. Product formation is, therefore, monitored by coupling the reaction to a non-enzymatic reaction that does yield a distinct signal. • eg. dihydroorate dehydrogenase

  41. Coupled Assay 3 • Enzymatic reaction of interest is paired with a 2nd enzymatic reaction which may be easily followed. • eg. Hexokinase • Caveats: • 1st enzyme MUST be rate limiting (product of 1st rxn must be immediately turned over by the second enzyme) • It is often difficult to match ideal conditions for 2 distinct enzymes • Inhibitors (or substrates) may affect both enzymes NAD+ NADH

  42. 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 measured at a specific time point on the linear range of the assay. Disadvantage: won’t notice deviations from linearity!

  43. Detection Methods • Spectrophotometry Abs = e  l  c (Beer’s law) vi = dc/dt = dAbs/dt (1/(e  l)) • Spectrofluorimetry • Radioactivity

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