Lectures in Enzymology

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)

Ni-men ha!

Lectures in Enzymology

My scientific interests:

• Kinetic studies of phosphotransferasesNucleoside diphosphate kinaseATP + 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)BiophysicsOrganic Chemistry –mechanism, stereochemistryPhysical Chemistry – kinetics, thermodynamicsStructural 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 mustunderstand 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 cellsAbout 10 mg from 3 kg of red blood cells Several isoenzymesWhat 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 dehydrogenaseIf 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 rapidlyYou get Sepharose!

Simple method for the preparation of spherical agarose and composite gel particlesPRESECAN E. ; PORUMB H. ; LASCU I. ;Inst. hygiene public health, Cluj-Napoca 3400, RomaniaJournal 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 denaturationand renaturation curves

100

150

200

250

300

350

0 1 2 3 4 5 6

Flu

ores

cenc

ein

tens

ity(a

rbitr

ary

units

)

[ Urea ], (M)

wt, denaturation

wt, renaturation

S120G, denaturation

S120G, renaturation

NDP kinase A denaturation/renaturation followedbythe fluorescence of Trp residues

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!

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!

Molecular biologist Biological Physicist

Your model is oversimplified and has nothing to do with biology!

Your model is too complicated and has no predictive power!

There are different ways to teach enzymology

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=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

Lectures in Enzymology: Recommended SoftwareYou 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 freeCurveExpert 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 chemicalmechanism and the software will calculete the concetration in function of time http://www.biochem.wustl.edu/cflab/message.htmlProtein 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 interactionshttp://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

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

Accelération (kcat)

HN

O

CN

O

OHOH

OP-O

O-

OO

OO

HN

O

N

O

OHOH

OP-O

O-

O O

HA

HN

O

N

O

OHOH

OP-O

O-

O O-A

CO2

knon = 2.8 x 10-16 s-1 kcat = 40 s-1 kcat /knon = 1.4 x 1017

t1/2 = 78 millions d’années t 1/2 = 18 millisecondes

VERY unstable intermediate

The interesting question is NOT how large is the rate acceleration, but how can the enzyme accelerate the reaction so much

OMP Decarboxylase (kcat/knon)

Orotidine 5’Phosphate decarboxylase

Fumaric acid 1 kg 8.5 mol 17.5 $ 2 $/molNH4OH 1 L 8.57 mol 16 $ 1.8 $/molL-Aspartic acid 1 kg 5.78 mol 119 $ 20.6 $/molDL-Aspartic acid 1 kg 73 $

1 mg…100 UI µmol/min 173*100*10-6 g 17 mg/min 24.9 g/24 h

A practical application (making money with the help of an enzyme) L-aspartate ammonia-lyase (aspartase).

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

1925 Briggs et Haldanestady-state

1915 Michelis-Menten (invertase)ES complex

1960 W. W. Clelandclassification of enzymatic reactionsmany useful developments od enzyme

kinetics

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 obtained1983 site-directed mutagenesis

Some history: The methods

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

Formal kinetics: the black box

Substrat Produit(s)

Substrat Produit(s)

Traps of formal kinetics….

C (t): the same kinetic equationRate-limiting step�the rate of the slowest step in a sequence�the value which we measure!

A B C110

[C] = [A]0 [1 + 1/(10-1)*(1*e-10*t – 10*e-1*t)]

A B C101

[C] = [A]0 [1 + 1/(1-10)*(10*e-1*t – 1*e-10*t)]

Enzymes are very EFFICIENT catalysts

Wild Type yeast: Yeast Trp94→AlaHighly active towards ethanol Activity 350-fold reducedWeakly active towards hexanol Activity 5-fold increased

HB HA

CH3O

Zn

Ser 48 (Yeast: Thr 48)

Nicotinamide

A’

C’

Leu 57

(Yeast: Trp 57)

Phe 93

(Yeast: Trp 94) B’

Enzymes are very SPECIFIC catalysts

alcohol dehydrogenase (EC 1.1.1.1)

HAHC

Pro-chiral

Méthodes d’étude du mécanisme catalytique de la chymotrypsine1. cinétique à l’état stationnaire

E + S � ES ES’ E + P2

P2INTERMEDIARE rapide

lente

rapide

lente

Méthodes d’étude du mécanisme catalytique de la chymotrypsine2. modification d’affinité

Modification chimique SERINE ACTIVE1950-1960

Di-isopropyl-fluorophosphate

Méthodes d’étude du mécanisme catalytique de la chymotrypsine3. Resolution de la structure 3D

D. Blow 1968

1. TRIADE CATALYTIQUE2. LA POCHE OXYANIONIQUE

Méthodes d’étude du mécanisme catalytique de la chymotrypsine4. 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 amino-terminal 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/

EC 1 OxidoreductasesEC 1.1 Acting on the CH-OH group of donorsEC 1.2 Acting on the aldehyde or oxo group of donorsEC 1.3 Acting on the CH-CH group of donorsEC 1.4 Acting on the CH-NH2 group of donorsEC 1.5 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 dehydrogenaseEC 1.1.1.2 alcohol dehydrogenase (NADP+)EC 1.1.1.3 homoserine dehydrogenaseEC 1.1.1.4 (R,R)-butanediol dehydrogenaseEC 1.1.1.5 acetoin dehydrogenaseEC 1.1.1.6 glycerol dehydrogenaseEC 1.1.1.7 propanediol-phosphate dehydrogenaseEC 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 dehydrogenaseEC 1.1.1.289 sorbose reductaseEC 1.1.1.290 4-phosphoerythronate dehydogenase

Classification des enzymes

Définitions, utilité

Enzymes = biocatalyseurs

protéines; (qq exemples d’ARN catalytiques = ribozymes )

Co-facteurs ions métaliques, Co-enzymesGroupement prostétiques

Avantages de la catalyse enzymatique(par rapport à la catalyse chimique)

Spécificité

Efficacité

Glucose oxydase

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 »

Pourqui étudier les enzymes ?

1. Ecrire la réaction catalysée par la cholinestérase (formules chimiques obligatoires!)

3a. Déduire l'équation de Michaelis qui décrit ce méc anisme. De quel type d’inhibition s’agît-il ?

L'enzymologie n'est pas une branche de l’algèbre !

Noté zéro si les passages n’ont pas été expliqués, même sil’équation finale est correcte !

Exemple de questions d’examen: