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Lectures in Enzymology

Lectures in Enzymology. Ni-men ha!. My name is Ioan LASCU Please send me an email for any question during these lectures! i.lascu@ ibgc.cnrs.fr I am Professor of Biochemistry at the University of Bordeaux (France). My scientific interests: Kinetic studies of phosphotransferases

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Lectures in Enzymology

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  1. Lectures in Enzymology Ni-men ha! My name is Ioan LASCU Please send me an email for any question during these lectures! i.lascu@ ibgc.cnrs.fr I am Professor of Biochemistry at the University of Bordeaux (France)

  2. My scientific interests: • Kinetic studies of phosphotransferases • Nucleoside diphosphate kinase • ATP + GDP  ADP + GTP • Folding and stability of proteins • Amyloid fibrils Lectures in Enzymology

  3. Bordeaux is the 6th town of France (about 600,000 people). It is famous for its wine and for its University.

  4. University of Bordeaux, France Lectures in Enzymology

  5. Lectures in Enzymology Sciences used for studying enzymes “Classical” biochemistry (cell metabolism) Biophysics Organic Chemistry –mechanism, stereochemistry Physical Chemistry – kinetics, thermodynamics Structural Biology Molecular Biology Bioinformatics But…… Studying enzymology may help understanding all that sciences

  6. I will try to explain the concepts in a simple way, so you may understand the basis of complex phenomena and may use these concepts for other situations. …..but….. « The trouble with simple things is that one must understand them very well » (Anonymous, cited by Donald T. Haynie, Biological Thermodynamics, Cambridge University Press 2001) Lectures in Enzymology Please interrupt me and ask if something is not clear (or we may discuss after the lectures)

  7. You may have success if a carreful analysis first, instead of working first…. Our example 1 Nucleoside diphosphate kinase from red blood cells About 10 mg from 3 kg of red blood cells Several isoenzymes What to do next? Other scientists separated the isoenzymes by complicated procedures and studied their kinetic properties

  8. At that time (1988-89) I was working in Romania and we were very poor and no good chromatographic equipment (in fact I fabricated myself columns, ion exchangers and affinity material) We made the following theoretical analysis: The several HEXAMERIC isoforms may be just the random association of two polypeptide chains, like lactate dehydrogenase If this is trus, the two kind of polypeptide, unfolded in urea, may be easily separated by simple ion exchange chromatography…. Calculated abondance of isoforms

  9. The designed experiment:

  10. The results: NDPK-B (basic) NDPK-A (acidic)

  11. How to make Sepharose using a paint blower? Agarose droplets are spherical, if you cool them rapidly You get Sepharose! Simple method for the preparation of spherical agarose and composite gel particles PRESECAN E. ; PORUMB H. ; LASCU I. ; Inst. hygiene public health, Cluj-Napoca 3400, Romania Journal of chromatography 1989, vol. 469, pp. 396-398

  12. You may have success if a carreful analysis first, instead of working first…. Our example 2 A B While teaching protein structure, one of the most proeminent properties of the native state is COOPERATIVITY. It is stabilized by a large number of weak interactions (Fig B rather than A). Experimentally, cooperativity translates by sigmoid denaturation and renaturation curves

  13. What would means a non-cooperative renaturation curve (here in red)? As we teach « native structure is cooperative », a non-cooperative curve would means that the structure is non-native! 350 300 250 200 150 100 0 1 2 3 4 5 6 NDP kinase A denaturation/renaturation followed by the fluorescence of Trp residues wt, denaturation wt, renaturation S120G, denaturation Fluorescence intensity (arbitrary units) S120G, renaturation [ Urea ], (M)

  14. Why studying the enzymes? “Because they exist” ….. basic knowledge Role in metabolism Enzymes are used in the analytical biochemistry to measure metabolite concentrations in complex misture (body fluids) Enzymes are used in the industrial biochemistry, to prepare useful molecules Most drugs are enzyme inhibitors ENZYMES FOR PLEASURE AND FOR PROFIT Lectures in Enzymology an over-view

  15. All lectures are available as PDF format in the following site http://lascu.free.fr/enzymology(be careful not to tape capital letters) Lectures in Enzymology: an over-view

  16. Lectures in Enzymology: an over-view There are three major TOOLS for studying enzyme mechanism Steady-state kinetics but enzymology is not a branch of the Algebra Structure Mutagenesis (site-directed or random)

  17. Your model is oversimplified and has nothing to do with biology! Your model is too complicated and has no predictive power! Molecular biologist Biological Physicist Lectures in Enzymology: an over-view Preparing lectures is useful for students (I hope) but for the professor, too: is the opportunity to think about the progress in enzymology since the last teaching! There are different ways to teach enzymology

  18. Lectures in Enzymology: an over-view Not all chapters of enzymology and not all classes of enzymes will be discussed here Examples will be from well studied pathways (glycolysis) Detailed description of proteases and phosphotransferases Once you have understood how the experimental data will be integrated into a theoretical model for one enzyme, it would be easy to do this for another enzyme

  19. Lectures in Enzymology: Recommended Books Lubert STRYER, Jeremy M. Berg, John L. Tymoczko BIOCHISTRY (necessary but not sufficient) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books&cmd=search&term=stryer Alan FERSHT STRUCTURE AND MECHANISM IN PROTEIN SCIENCE: A GUIDE TO ENZYME CATALYSIS AND PROTEIN FOLDING W. H. Freeman, New York 1999

  20. You can fing excellent software for free: some people spent time and energy for the colleagues Software for fiting experimental data Kaleidagraph Costly, but the DEMO version is free CurveExpert A very good freeware, excellent algoritm Monte Carlo Simulation : Chemical Kinetics Simulator (CKS). Can be download at: https://www.almaden.ibm.com/st/computational_science/ck/msim/) Simulation and fitting: KINSIM et FITSIM. Developped by C. Frieden; you write the chemical mechanism and the software will calculete the concetration in function of time http://www.biochem.wustl.edu/cflab/message.html Protein structure RASMOL. Coloured and easy to use (http://mc2.cchem.berkeley.edu/Rasmol/v2.6/) SwissPDBViewer Less good as graphics, but excellent for studying biomolecular interactions http://www.expasy.org/spdbv/text/download.htm Lectures in Enzymology: Recommended Software

  21. Drawing chemical structures ISIS Draw 2.4 Very easy to use www.mdli.com/download/ Bitmap images Irfan View (http://www.irfanview.com/) or Paint of Microsoft A navigator (I use Firefox) A software for reading pdf documents. Foxit Reader is fast and free! Lectures in Enzymology: Recommended Software

  22. Specificity --- the ability of enzymes to discriminate between a substrate and a competing molecule. High specificity --- functional groups in the active site of enzyme arranged optimally to form a variety of weak interactions with a given substrate in the transition state

  23. Accelération (kcat) Logarithmic scale of kcat and knon values for some representative reactions at 25 °C. The length of each vertical bar represents the rate enhancement by ADC ) arginine decarboxylase; 25 ODC ) orotidine 5¢-phosphate decarboxylase;23 STN ) staphylococcal nuclease;17 GLU ) sweet potato â-amylase;13 FUM ) fumarase;21 MAN ) mandelate racemase;22 PEP ) carboxypeptidase B;14 CDA ) E. coli cytidine deaminase;30 KSI ) ketosteroid isomerase;23 CMU ) chorismate mutase;19 CAN ) carbonic anhydrase.23

  24. Orotidine 5’Phosphate decarboxylase OMP Decarboxylase (kcat/knon) VERY unstable intermediate knon = 2.8 x 10-16 s-1kcat = 40 s-1kcat /knon = 1.4 x 1017 t1/2 = 78 millions d’années t1/2 = 18 millisecondes The interesting question is NOT how large is the rate acceleration, but how can the enzyme accelerate the reaction so much

  25. A practical application (making money with the help of an enzyme) L-aspartate ammonia-lyase (aspartase). Fumaric acid 1 kg 8.5 mol 17.5 $ 2 $/mol NH4OH 1 L 8.57 mol 16 $ 1.8 $/mol L-Aspartic acid 1 kg 5.78 mol 119 $ 20.6 $/mol DL-Aspartic acid 1 kg 73 $ 1 mg…100 UI µmol/min 173*100*10-6 g 17 mg/min 24.9 g/24 h

  26. Another practical application (making economies) If you needs large amounts of TDP (thymidine 5’-diphosphate) 25 mg (62 µmol)……240$, or 3.87 $/µmol Phosphorylate the TMP which costs 0.13 $/µmol!!

  27. Some history: The methods 1915Michelis-Menten   (invertase) ES complex 1925 Briggs et Haldane stady-state 1960 W. W. Cleland classification of enzymatic reactions many useful developments od enzyme kinetics

  28. 1915 Enzymatic kinetics (Michaelis) The physical nature of enzymes was unknown (ill- defined “colloids”) 1928 Urease – crystallization of an enzyme. This was an essential step: enzymes are homogeneous moleculas which can be studied by chemical means 1955 Sanger: first sequence of a small protein (insuline) 1968 X-ray structure of an enzyme: Blow  (chymotrypsin) 1950-1980 affinity labeling for identifying the active-site residues (now in disuse)1978 Gene cloning: recombinant enzymes could be obtained 1983 site-directed mutagenesis Some history: The methods

  29. Substrat Produit(s) Substrat Produit(s) Formal kinetics: the black box 1a. Can be used independently of enzyme structure 1b. No hint on enzyme structure 2. Can be compatible with a mechanism, but is not a proof

  30. 10 1 A B C [C] = [A]0 [1 + 1/(10-1)*(1*e-10*t – 10*e-1*t)] 1 10 A B C [C] = [A]0 [1 + 1/(1-10)*(10*e-1*t – 1*e-10*t)] Traps of formal kinetics…. C (t): the same kinetic equation • Rate-limiting step • the rate of the slowest step in a sequence • the value which we measure!

  31. Enzymes are very EFFICIENT catalysts

  32. Enzymes are very SPECIFIC catalysts Zn A’ Ser 48 (Yeast: Thr 48) C’ Nicotinamide alcohol dehydrogenase (EC 1.1.1.1) Phe 93 (Yeast: Trp 94) B’ HA HC Leu 57 (Yeast: Trp 57) Wild Type yeast: Yeast Trp94Ala Highly active towards ethanol Activity 350-fold reduced Weakly active towards hexanol Activity 5-fold increased

  33. Pro-chiral

  34. rapide lente lente rapide Méthodes d’étude du mécanisme catalytique de la chymotrypsine1. cinétique à l’état stationnaire E + S  ES ES’ E + P2 P2 INTERMEDIARE

  35. Méthodes d’étude du mécanisme catalytique de la chymotrypsine2. modification d’affinité Modification chimique SERINE ACTIVE 1950-1960 Di-isopropyl-fluorophosphate

  36. Méthodes d’étude du mécanisme catalytique de la chymotrypsine3. Resolution de la structure 3D D. Blow 1968 1. TRIADE CATALYTIQUE 2. LA POCHE OXYANIONIQUE

  37. Méthodes d’étude du mécanisme catalytique de la chymotrypsine4. La mutagenèse dirigée

  38. Classification des enzymes La nomenclature EC (EC est le sigle de Enzyme Commission numbers, la Commission des enzymes) est une classification numérique des enzymes, basée sur la réaction chimique qu'elles catalysent. En tant que système de nomenclature des enzymes, chaque numéro EC est associé à un nom recommandé pour l'enzyme correspondante. Chaque code d'enzyme consiste en les lettres majuscules « EC » suivies de quatre nombres séparés par des points. Ces nombres représentent chacun une étape dans la précision de la classification de l'enzyme. Par exemple, l'enzyme tripeptide aminopeptidase a le code EC 3.4.11.4 qui est construit comme suit : 3 signifie une hydrolase (enzymes qui utilisent l'eau pour détruire une autre molécule), 3.4 signifie hydrolases agissant sur des liens peptidiques, 3.4.11 implique celles qui détachent un acide aminé amino-terminal d'un polypeptide et 3.4.11.4 implique celles qui détachent cet acide aminé amino-terminal d'un tripeptide.

  39. Classification des enzymes Le niveau supérieur de cette classification est * EC 1 Oxydoréductases : catalysent les réactions d'oxydo-réduction * EC 2 Transférases : transfèrent un groupement fonctionnel (par exemple un groupe méthyle ou phosphate) * EC 3 Hydrolases : catalysent l'hydrolyse de diverses liaisons * EC 4 Lyases : brisent diverses liaisons par d'autres procédés que l'hydrolyse et l'oxydation * EC 5 Isomérases : catalysent les réactions d'isomérisation dans une simple molécule * EC 6 Ligases : joignent deux molécules par des liaisons covalentes La nomenclature complète peut être vue à l'adresse http://www.chem.qmul.ac.uk/iubmb/enzyme/

  40. Classification des enzymes EC 1 Oxidoreductases EC 1.1 Acting on the CH-OH group of donors EC 1.2 Acting on the aldehyde or oxo group of donors EC 1.3 Acting on the CH-CH group of donors EC 1.4 Acting on the CH-NH2 group of donors EC 1.5 Acting on the CH-NH group of donors EC 1.6 Acting on NADH or NADPH EC 1.1.1.1 alcohol dehydrogenase EC 1.1.1.2 alcohol dehydrogenase (NADP+) EC 1.1.1.3 homoserine dehydrogenase EC 1.1.1.4 (R,R)-butanediol dehydrogenase EC 1.1.1.5 acetoin dehydrogenase EC 1.1.1.6 glycerol dehydrogenase EC 1.1.1.7 propanediol-phosphate dehydrogenase EC 1.1.1.8 glycerol-3-phosphate dehydrogenase (NAD+) EC 1.1.1.9 D-xylulose reductase EC 1.1.1.287 D-arabinitol dehydrogenase (NADP+) EC 1.1.1.288 xanthoxin dehydrogenase EC 1.1.1.289 sorbose reductase EC 1.1.1.290 4-phosphoerythronate dehydogenase EC 1.1.1 With NAD or NADP as acceptor EC 1.1.2 With a cytochrome as acceptor EC 1.1.3 With oxygen as acceptor EC 1.1.4 With a disulfide as acceptor EC 1.1.5 With a quinone or similar compound as acceptor EC 1.1.99 With other acceptors

  41. Définitions, utilité Enzymes = biocatalyseurs protéines; (qq exemples d’ARN catalytiques = ribozymes ) Co-facteurs ions métaliques, Co-enzymes Groupement prostétiques Avantages de la catalyse enzymatique (par rapport à la catalyse chimique) Spécificité Efficacité

  42. Glucose oxydase

  43. Pourqui étudier les enzymes ? Etudes fondamentales "parce'qu-elles existent"= relation structure-activité Biologie - métabolisme Biochimie analytique (glucose, anomers) Biochimie industrielle (glucose isomérase) 60°C, Co2+ Médecine (médicaments = inhibiteurs) « drug design »

  44. L'enzymologie n'est pas une branche de l’algèbre ! Exemple de questions d’examen: 1. Ecrire la réaction catalysée par la cholinestérase (formules chimiques obligatoires!) 3a. Déduire l'équation de Michaelis qui décrit ce mécanisme. De quel type d’inhibition s’agît-il ? Noté zéro si les passages n’ont pas été expliqués, même si l’équation finale est correcte !

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