Lectures in Enzymology
Ni-men ha!
My name is Ioan LASCU Please send me an email for any question during these lectures!
[email protected] I am Professor of Biochemistry at the University of Bordeaux (France)
Lectures in Enzymology
My scientific interests: • Kinetic studies of phosphotransferases Nucleoside diphosphate kinase ATP + GDP ADP + GTP • Folding and stability of proteins • Amyloid fibrils
Bordeaux is the 6th town of France (about 600,000 people). It is famous for its wine and for its University.
Lectures in Enzymology University of Bordeaux, France
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
Lectures in Enzymology
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)
Please interrupt me and ask if something is not clear (or we may discuss after the lectures)
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
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
The designed experiment:
The results: NDPK-B (basic) NDPK-A (acidic)
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
You may have success if a carreful analysis first, instead of working first…. Our example 2 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). A
B
Experimentally, cooperativity translates by sigmoid denaturation and renaturation curves
What would means a non-cooperative renaturation curve (here in red)? As we teach « native structure is cooperative », a noncooperative curve would means that the structure is non-native!
Fluorescence intensity (arbitrary units)
350 NDP kinase A denaturation/renat uration followed by the fluorescence of Trp residues
300
wt, denaturation
250 wt, renaturation 200
S120G, denaturation S120G, renaturation
150
100 0
1
2
3 [ Urea ], (M)
4
5
6
Lectures in Enzymology an over-view 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 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)
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 Your model is oversimplified and has nothing to do with biology!
Molecular biologist
Your model is too complicated and has no predictive power!
Biological Physicist
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
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=se arch&term=stryer
Alan FERSHT STRUCTURE AND MECHANISM IN PROTEIN SCIENCE: A GUIDE TO ENZYME CATALYSIS AND PROTEIN FOLDING W. H. Freeman, New York 1999
Lectures in Enzymology: Recommended Software 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 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!
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
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 âamylase;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
OMP Decarboxylase (kcat/knon) Orotidine 5’Phosphate decarboxylase
O
O
HN
HN O O
O -O
P
N
-O
O
O
P
O
O-
O
N
-A
O
O-
CO2 OH
O
O
C
O
OH
OH
OH
HN O
O
VERY unstable intermediate
-O
P
O
N O
HA
OOH
knon = 2.8 x 10-16 s-1
kcat = 40 s-1
t1/2 = 78 millions d’années
OH
kcat /knon = 1.4 x 1017
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
A practical application (making money with the help of an enzyme) L-aspartate ammonia-lyase (aspartase).
Fumaric acid NH4OH L-Aspartic acid DL-Aspartic acid
1 mg…100 UI 173*100*10-6 g
1 kg 1L 1 kg 1 kg
8.5 mol 8.57 mol 5.78 mol
17.5 $ 2 $/mol 16 $ 1.8 $/mol 119 $ 20.6 $/mol 73 $
µmol/min 17 mg/min 24.9 g/24 h
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!!
Some history: The methods 1915 Michelis-Menten (invertase) ES complex
1925 Briggs et Haldane stady-state
1960 W. W. Cleland classification of enzymatic reactions many useful developments od enzyme kinetics
Some history: The methods 1915 Enzymatic kinetics (Michaelis) The physical nature of enzymes was unknown (illdefined “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
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
Substrat
Substrat
Produit(s)
Produit(s)
Traps of formal kinetics….
10
A
1
B
C
[C] = [A]0 [1 + 1/(10-1)*(1*e-10*t – 10*e-1*t)] 1
A
10
B
C
[C] = [A]0 [1 + 1/(1-10)*(10*e-1*t – 1*e-10*t)] C (t): the same kinetic equation Rate-limiting step the rate of the slowest step in a sequence the value which we measure!
Enzymes are very EFFICIENT catalysts
Enzymes are very SPECIFIC catalysts alcohol dehydrogenase (EC 1.1.1.1) Zn
Phe 93 (Yeast: Trp 94)
A’
B’ H3C O HB HC C’
Ser 48 (Yeast: Thr 48)
HA HA Nicotinamide
Leu 57 (Yeast: Trp 57)
Wild Type yeast: Highly active towards ethanol Weakly active towards hexanol
Yeast Trp94→Ala Activity 350-fold reduced Activity 5-fold increased
Pro-chiral
Méthodes d’étude du mécanisme catalytique de la chymotrypsine 1. cinétique à l’état stationnaire
E+S
ES
ES’
E + P2
P2 INTERMEDIARE
rapide lente
lente rapide
Méthodes d’étude du mécanisme catalytique de la chymotrypsine 2. modification d’affinité
Modification chimique SERINE ACTIVE 1950-1960
Di-isopropyl-fluorophosphate
Méthodes d’étude du mécanisme catalytique de la chymotrypsine 3. Resolution de la structure 3D
D. Blow 1968
1. TRIADE CATALYTIQUE 2. LA POCHE OXYANIONIQUE
Méthodes d’étude du mécanisme catalytique de la chymotrypsine 4. La mutagenèse dirigée
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 aminoterminal d'un polypeptide et 3.4.11.4 implique celles qui détachent cet acide aminé amino-terminal d'un tripeptide.
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/
Classification des enzymes EC 1 EC 1.1
Oxidoreductases Acting on the CH-OH group of donors
EC 1.2 EC 1.3 EC 1.4 EC 1.5
Acting on the aldehyde or oxo group of donors Acting on the CH-CH group of donors Acting on the CH-NH2 group of donors Acting on the CH-NH group of donors
EC 1.6
Acting on NADH or NADPH
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
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
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
Glucose oxydase
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 »
L'enzymologie n'est pas une branche de l’algèbre ! Exemple de questions d’examen: 1. Ecrire la réaction chimiques obligatoires!)
catalysée
par
la
cholinestérase
(formules
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 !
