ORIGIN OF THE CATALYTIC POWER OF ENZYMES

1. 2. 3. 4. 5. 6.

General acid-base catalysis Covalent catalysis Metal ion catalysis Electrostatic catalysis Intramolecular catalysis: Proximity and orientation Preferential binding by enzyme of the Transition state over the substrate(s)

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

• Uncatalyzed reactions are slow because they must proceed through highly unfavorable transition states. • Catalysts speed the rate of a reaction by lowering the activation energy barrier for that reaction. Enzymes, as discussed in the previous lecture, are designed to preferentially stabilize the transition state over the substrate. In so doing, they greatly increase the rate of a reaction.

ORIGIN OF THE CATALYTIC POWER OF ENZYMES • Homolytic cleavage: radical species are unstable and not readily formed. Mechanisms involving free radicals are less common in enzymology. C

H

C

H

• Heterolytic cleavage: One atom retains both electrons. This usually leaves a charge on one or both products. C

H

C

H

proton transfer

carbanion

C

H

C

carbocation

H

hydride transfer

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

• Arrows denote the movement of a pair of electrons • They always move in one direction: electronegative to electropositive (from nucleophiles to electrophiles)

i.e. Schiff base formation H+ R

NH2

+

C R1

R2

R2

R2 O

R

N

C

H

R1

R

OH H+

N

C

H

R1

+ H2 O

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

General acid-base catalysis The uncatalyzed hydrolysis of an acetal leads to a transition state in which positive and negative charges develop across the C—O bond. This charge separation is quite unfavorable and leads to a very slow rate of reaction.

O

O OR

δ+

δ−

OR

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

General acid-base catalysis The general acid catalyzed hydrolysis of an acetal stabilizes the developing negative charge on the oxygen of the acetal by transferring a proton from the general acid catalyst.

O

O OR

H

A

δ+

OR H

A

δ−

Partial proton transfer from a general acid catalyst lowers the free energy of activation by stabilizing the developing negative charge in the transition state.

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

General acid-base catalysis The uncatalyzed attack of water on an ester leads to a transition state in which positive charge develops on the attacking water molecule and negative charge develops on the carbonyl oxygen. These developing charges are unfavorable in the transition state. O R'

C

O δ− OR

R'

C

+ O H

O H

H

δ+

H

OR

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

General acid-base catalysis The general base catalyzed attack of water on an ester allows the developing positive charge on the water oxygen to be stabilized by the transfer of its proton to the general base catalyst. O R'

δ−

C

O

OR

R'

C

+ O H

O H

H

OR

δ−

H

δ+ δ+

NH2 NH2

B

B

Partial proton transfer to a general base catalyst lowers the free energy of activation by stabilizing the developing positive charge in the transition state.

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

General acid-base catalysis • Acid-base catalysis is termed “general” to distinguish it from specific acid-base catalysis. In specific acidbase catalysis, the catalyst is the proton or hydroxide ion itself. Specific acid-base catalysis cannot be effective with enzyme which should act at neutral pH. • Concerted general acid-base catalysis occurs when a reaction is simultaneously subject to both general acid and general base catalysis.

ORIGIN OF THE CATALYTIC POWER OF ENZYMES Acid/base catalysis: (a) specific acid-base catalysis general acid-base catalysis H2O

O H3C

N

N

O

+

H3C

HN

N

O

HO H N

NH

(b) general acid-base catalysis This reaction accelerated by imidazole. Usually increasing concentration of product(imidazole) will decrease the rate. However, imidazole help to extract H+from water molecules in T.S.

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

RNase A

ORIGIN OF THE CATALYTIC POWER OF ENZYMES

Enolase general base catalyst Lys 3 4 5

HOOC-Glu 2 1 1

general acid catalyst

H

H2 O

Lys 345

H

H2 N:

-

OH

O

O

P

H O O-

O

O

+ O

H2 C

H2 NH+

-

O

-

O O-

2-phosphoglycerate

OOC-Glu 211

P O

O

-

-

phosphoenolpyruvate

HOOC-Glu 211 Lys 345

H H

+

OH

H2 NH

O -

O O-

O O

P O

-

-

ene-diolate intermediate

Covalent catalysis - Schiff Base formation • A Schiff base may form from the condensation of an amine with a carbonyl compound H+ R

NH2

+

C R1

R2

R2

R2 O

R

N

C

H

R1

R

OH H+

N

C

H

R1

+ H2 O

• The Schiff base (protonated at neutral pH) acts as an electron sink that greatly stabilizes negative charge that develops on the adjacent α-carbon.

Covalent catalysis - Schiff Base formation Uncatalyzed decarboxylation of acetoacetate

O H3 C

C

H2 C C

O

O H3 C O

H+

C

O

O CH2

+

C

O

a highly unstable enolate intermediate ∴ a very unfavorable reaction

H3 C

C

CH3

Example: Acetoacetate Decarboxylase Schiff base catalyzed decarboxylation of acetoacetate H+ O H3 C

C

H2 C

CO 2

H2 O

O H3 C

C O

C

H2 C

H2 O

+ H+ H3 C

C O

NH H+

O C

CH2

NH E

E

a stable intermediate

NH2 E

H+ O H3 C

C

NH2 E

H2 O H3 C

CH3

C NH

H+

H2 O

E

CH3

Evidence for Schiff base intermediate Enzymes that form Schiff base intermediates are typically irreversibly inhibited by the addition of sodium borohydride (Na+ BH4–). Borohydride reduces the Schiff base and “traps” the intermediate such that it can no longer be hydrolyzed to release the product from the enzyme: H H3 C

C NH E

CH3

BH3 H3 C

H C

CH3

+ BH3

NH E

This is often used as evidence for a mechanism involving an enzymelinked Schiff base intermediate. NaBH4 reduces both the Schiff base and the carbonyl; For a specific reduction of Schiff base, but not of aldehyde/cetone use NaBH3CN

Covalent catalysis - Pyridoxal phosphate H

O C

H

O O O

P

O

O N

CH3

H

coenzyme PLP R

R

NH2

H H C

O P

O

H

O

H

O O

N C

O

Schiff Base

O O

P

O

O N

O N H

CH3

H

CH3

Example: Arginine Decarboxylase An extremely unstable intermediate

Spontaneous reaction O

O-

H

C

(-)C

CH R

H R

HC

+ CO2

NH2

NH2 H

NH2

R

-17 s-1 kuncatalyzed: ~ 2 x 10–17 –1

k = 2 × 10

s

-OH

HO

NH H2 C

R=

H2 C

H2 C

H N

C

Arginine decarboxylase + Enz-PLP

-O

O-

O C HC

O

R

NH2

NH2

A stable intermediate BH

H

C

C

CH R

N

H R

+ CO2

HC

+ Enz-PLP R

NH2

+HN

O-

2-O PO 3

O-

2-O PO 3

N H N H+

PLP alone: k = 3.9 × 10–6 s–1 Arg decarboxylase: = 1375 kcat =kcat 1375 s-1 s–1 kPLP/knon = 2 × 1011 kcat/knon = 7 × 1019

Covalent catalysis - Thiamine pyrophosphate NH2 H2 C

H C N

S

N H3 C

O H2 C

N

H2 C

O

P

O O

O

P

OH

O

coenzyme TPP

H+

H C R

N

C R

S

R

1

H+

N

S

R

1

The positive charge on the nitrogen promotes ionization of the C-2 carbon by electrostatic stabilization. The ionized carbon is a potent nucleophile.

Example: Pyruvate Decarboxylase pyruvate

CO 2 H+

O C O

O

O

O C

+ HO

C CH3

C N

S

R1

S

C

S

C

HO

C

C

N

CH3

H+

+ H+

R1

N

CH3 R

R

R1

R

acetaldehyde

H

H

H

+ O

O

C

C CH3

C N R

R1

H+

S

H+ R1

CH3

S C N R

Covalent catalysis - Nucleophilic catalysis Common nucleophlic groups on enzymes & example enzymes: Serine hydroxyl S

SH

Serine proteases Cholinesterases Esterases

+ H+

Cysteine thiol

Thiol proteases G3P dehydrogenase O

OH

+ H+

Lysine amino group

PLP-dependent enzymes acetoacetate decarboxylase

NH2 + H+

NH3

Histidine imidazole H

H

N

phosphoglycerate mutase succinyl-CoA synthetase

N + H+

N H

N

Example: Serine Proteases Asp

Asp

O

O

H

CH2

N N

H

S er

C

Ser

C

As p

His

His O

O

H

N

O

Se r

C

CH2

N

His

O

O

H

CH2

N

O

N

O

H R1

R N

H

C

H

R

R1

N

R1

C

H

O

N

R C

H

O

O

H2 O

R 1NH2 As p

His

As p

O

O

As p

His

Ser

C

Ser

C H

CH2

N N

H

O

O

O

H

CH2

N N

O

His Ser

C O

O

H

CH2

N N

O

H H

R R O

O H

H

C

O

C O

O

H

R C O

Metal ion catalysis • Two classes: • metalloenzymes (Fe2+, Fe3+, Cu2+, Zn2+, Mg2+, Mn2+, or Co3+) The metal ion does not dissociate at every catalytic cycle. Tightly bound to the enzyme • metal-activated enzymes (Na+, K+, Mg2+, or Ca2+) Dissociate at every catalytic cycle • Participate in reactions: – By binding substrates – By mediating oxidation-reduction reactions – By electrostatically stabilizing or shielding negative charges

Metal ion catalysis • Can make a reaction center more susceptible to receiving electrons because it can stabilize a developing negative charge on a transition state. (electrophilic catalyst)

Metal ion catalysis

• Can stabilize developing negative charge on a leaving group, making it a a better leaving group.

(electrophilic catalyst)

Metal ion catalysis • Can increase the rate of a hydrolysis reaction by forming a complex with water, thereby increasing water’s acidity.

Example: β-lactamase (B. cereus)

3. Catalyse par des ions métaliques

3. Catalyse par des ions métaliques Zn2+ ion decreases the pHa of water 108-fold KH20

OH- + H+

OH2 KH2O

KHOKZn-H20

Zn2+ -OH2

Zn2+-OH- + H+

This is a thermodynamic cycle! Zn2+ ion binds stronger the OHthan H2O. As a direct consequence, KZn-H20 >> KH20

Electrostatic Catalysis • Charge distributions about the active sites of enzymes are arranged so as to stabilize the transition states of the catalyzed reactions • These charge distributions may also serve to guide polar substrates towards their binding sites so that the rates of these enzymatic reactions are greater than their apparent diffusioncontrolled limits – i.e. Superoxide dismutase (kcat/Km = 2.0 × 109 M–1 s–1) Functions faster than the diffusion limit! 4 O 2 - + 4 H+

2 H2 O 2 + 2 O 2

Example: Superoxide Dismutase

Zhou et al. (1997) PNAS 23, 12372.

Electrostatic Catalysis • The local dielectric constant of the active site of an enzyme probably resembles more of that for an organic solvent than for water. Thus the pKs of amino acid side chains in proteins may vary by several units from their nominal values because of the proximity of charged groups.

Catalysis by Approximation (Entropic Contribution) • Rate enhancements result from positioning the substrate in close proximity to the reacting group(s) (or, in multi-substrate enzymes, positioning two or more substrates in close proximity to each other). • Molecules react most readily if they are properly oriented relative to each other. It has been estimated that properly orientating substrates can increase reaction rates by a factor of up to ~100. • Enzymes bind substrates in a manner that both immobilizes them and aligns them so as to optimize their reactivities.

2. Orientation et proximité des substrats La catalyse enzymatique évite la perte d’entropie

A + B

A

B

AB‡

AB‡

2. Orientation et proximité des substrats

La formation du complexe perte de degrées de liberté transationelle et rotationnelle du substrat dislocation de N molécule d’eau ∆S > 0 Interactions enthalpiques favorables ∆H < 0 ∆G = ∆H - T∆S

S = R ln W

∆S < 0

2. Orientation et proximité des substrats

k = 35 M-1 s-1

k = 840 s-1

Be carreful when comparing the rate constant of a first order reaction with the rate constant of a second order reaction: Just multiply the second order rate constant by 1.0 M ! It will become a pseudo-order rate constant.

2. Orientation et proximité des substrats

2 reactants in solution

The 2 reactive groups belongs to the same molecule The 2 reactive groups belongs to the same molecule, which is sterically blocked

The concentration calculated from the ratio of the reaction rates is called EFFECTIVE CONCENTRATIONS. The rates constants are compared with the reaction (a) at 1 M acetate!

Catalysis by Approximation (Entropic Contribution) • In binding their substrate(s), enzymes freeze out the relative rotational and translational motions of the substrate(s) and reacting group(s). This can result in significant rate enhancemenst (up to ~ 107) • There is, however, a significant entropic cost associated with this loss of translational and rotational freedom. It is paid for by the very favorable enthalpy associated with substrate binding.

S06a. Etat natif: Propriétés globales, coopérativité Les interactions non-covalentes sont faibles dans l’eau……. mais ont une contribution coopérative (ab > a + b)

Comparer avec la réaction AB A+B

effective molarity (effective concentration) The ratio of the first-order rate constant of an intramolecular reaction involving two functional groups within the same molecular entity to the second-order rate constant of an analogous intermolecular elementary reaction. This ratio has the dimension of concentration. The term can also apply to an equilibrium constant. IUPAC Compendium of Chemical Terminology 2nd Edition (1997)

5. Stabilization de l’état de transition An enzyme has a structure closely similar to that found for antibodies, but with one important difference, namely, that the surface configuration of the enzyme is not so closely [complementary] to its specific substrate as is that of an antibody to its homologous antigen, but is instead complementary to an unstable molecule with only transient existence—namely, the 'activated complex' for the reaction that is catalyzed by enzyme Linus Pauling, 1948 Linus Pauling (1901-1994) “invented” the resonance theory and the α-helix

Comparing a spontaneous reaction (1) with the same reaction, catalyzed by a enzyme (2) 1.

2. A thermodynamic cycle: if S‡ binds strionger than S to the enzyme E, KE‡ should be lower (lower activation energy)

Transition State Theory and Thermodynamic Cycles A popular model for comparing the rates of catalyzed and uncatalyzed reactions, that is, the thermodynamic cycle as set up in Scheme 1, which compares a transfermation of a reactant S in solution through its transition state (S‡) with the same reaction catalyzed by an enzyme from its ground state (ES) to its transition state (ES‡). The solution reaction is characterized by the reaction rate knon, and the enzyme-catalyzed reaction is characterized by the Michaelis-Menten constants kcat and Km. The value of KTS is generally significantly smaller than the dissociation constant for the substrate from the enzyme, Ks, leading to a large and favorable free energy of binding for the enzyme and S‡.

2 experimental proofs that enzyme bind indeed stronger the transition state than substrate(s): A. Transition state analogs are strong enzyme inhibitors B. Injecting transition state analogs to animals, some antibodies secreted are catalytic.

5. Stabilization de l’état de transition Transition state structure is a hypothesis: its life-time is so short that it cannot be studied by any physical method Hypothesis based on the chemical knowledge The Hammond postulate: • Related species that are similar in energy are also similar in structure. The structure of a transition state resembles the structure of the closest stable species. • Transition state structure for endothermic reactions resemble the product. • Transition state structure for exothermic reactions resemble the reactants.

5. Stabilization de l’état de transition Hammond postulated that in highly exothermic reactions (left) the transition state (Ts) is structurally similar to the reactant (R),

In highly endothermic reactions (right) the product (P) is a better model of the transition state.

5a. Analogues de l’état de transition sont des inhibiteurs

E-L-Pro

Figure 1 Gibbs free energy profile for the enzymatic reaction.

3/20/2015

E-D-Pro

Figure 2 Schematic representation of the residues interacting with the QM subregion (shown in green).

Published in: Marco Stenta; Matteo Calvaresi; Piero Alto; Domenico Spinelli; Marco Garavelli; Andrea Bottoni; J. Phys. Chem. B 2008, 112, 10573/20/2015 1059. DOI: 10.1021/jp7104105 Copyright © 2008 American Chemical Society

Transition state structures and transition state analogue inhibitors for PNPs.

Schramm V L J. Biol. Chem. 2009;284:32201-32208

Wilson, D. K. et. al. Atomic Structure of Adenosine Deaminase Complexed with a Transition-State Analog: Understanding Catalysis and Immunodeficiency Mutations. (1991.) Science 252 (5010). 1278.

5a. Analogues de l’état de transition sont des inhibiteurs Adenosine 5’-monophosphate (AMP) deaminase: – Involved in the purine nucleotide cycle – Since adenosine is a known cardioprotective agent and AMP deaminase shunts adenine nucleotides away from adenosine, AMP deaminase inhibitors could have therapeutic value in speeding recovery from heart attacks.

Zn 2+ OH

Zn 2+

Zn 2+

NH2

OH N

N

P O

N

N

O O O

N

N

O H H

H

OH

H

AMP

H

R

N

N

N N

HO

NH2

N

+ NH3 +

R

IMP

N

5a. Analogues de l’état de transition sont des inhibiteurs AMP Deaminase (KTx = 1.3 × 10–17 M)

Kd

Kd

5a. Analogues de l’état de transition sont des inhibiteurs Fluoroaluminates

AlF3

ADP

Ser phosphorylée

Le site actif de la Protéine Kinase - A Fichier PDB 1L3R (se trouve dans le dossier RCE cours)

Fig. 4. Crystal structures of bovine purine nucleoside phosphorylase (PNP) with substrate analogues (A), transition state analogue (B) and products (C) bound at the catalytic sites. Conversion from substrate to transition state analogue forms new hydrogen bonds and closer atomic contacts (red in B) and one relaxed distance that contributes to the nucleophilicity of phosphate (blue in B). In the product complex, multiple atomic distances relax to form weaker contacts (blue in C).

Archives of Biochemistry and Biophysics Volume 433, Issue 1, 1 January 2005, Pages 13-26 Enzymatic transition states: thermodynamics, dynamics and analogue design Vern L. Schramm

Substrate alignment Arginine kinase

5a. Analogues de l’état de transition sont des inhibiteurs Inhibiteurs analogues de l’état de transition utilisés comme médicaments Maladie

Enzyme cible

Affinité

Alzheimer’s disease SIDA Cancer

γ-secretases protease 5’-methylthioadenosine human glyoxylase I nucleoside phosphorylase nucleoside hydrolase

< 1 µM 10 – 100 pM 200 pM 1 nM 20 pM 2 nM

Lymphoma Parasitic infections

5b. Anticorps catalytiques Crystal Structure of a Catalytic Antibody with a Serine Protease Active Site. Zhou, G; Guo, Jincan; Huang, Wei; Fletterick, Robert; Scanlan, Thomas Science. 265(5175):1059-1064, August 19, 1994.

1EAP Figure 1 . (A) Amino acid ester hydrolysis reaction catalyzed by antibody 17E8 and structure of norleucine phosphonate hapten (4) used to elicit the antibody 17E8. The hapten is designed to mimic the transition state (3) leading to the tetrahedral hydrolysis intermediate. The antibody catalyzes the hydrolysis of both norleucine (1) and methionine (2) phenyl esters. (B) The pH rate profile for 17E8-catalyzed hydrolysis of the norleucine phenyl ester 1; the methionine ester 2 gives a similar pH rate profile. Plots of kcat over KM for 1 and 2 are similarly bell-shaped. The data are fit to a rate equation (r equals 0.995) derived from the equilibrium relation where the singly protonated antibody species (IgGH) is the most catalytically active state [11]. The pKa values of 9.1 and 10.0 are calculated from the fit.

Figure 5 . A closeup view of the 17E8 active site in the crystal structure (A), in the proposed reactive conformation (B), and superimposition of the 17E8 active site reactive conformation with the active site of trypsin complexed to bovine pancreatic trypsin inhibitor (C). (A) The backbone of the antibody is shown as a ribbon with the same color code and view as in Fig. 3. The side chain atoms of the important amino acids are shown in ball-and-stick format and color-coded as: C, green; O, red; N, blue; H, white. The hapten 4 is shown in yellow as a ball-and-stick model with the phosphorous atom in red. SerH99 accepts a hydrogen bond from the donor TyrH101 at neutral pH. (B) At pH 9.5 this hydrogen bond is removed and SerH99 is free to rotate about the C alpha-C beta bond. A 180 degrees rotation about C alpha-C beta of SerH99 would orient the O gamma of SerH99 for nucleophilic attack at the substrate carbonyl carbon to form an acyl intermediate. The nucleophilicity of the SerH99 hydroxyl would be enhanced by a hydrogen bond to the N epsilon of HisH35 (see Table 2. The N epsilon of LysH97 resides on the opposite face of the substrate carbonyl and functions electrostatically to stabilize the developing negative charge at the carbonyl oxygen. (C) Superimposition of the active site reactive conformation of 17E8 and the active site structure of trypsin complexed with BPTI. The catalytic residues are on the right side with backbone and side chain atoms of trypsin in white and pink, respectively, and backbone and the side chain atoms of 17E8 in yellow and red, respectively. The trypsin active site contains a Ser-HisAsp catalytic triad whereas the catalytic antibody active

5b. Anticorps catalytiques Les premiers anticorps catalytiques: réaction estérase

H

H

H

O

H

H

O R

O

O

+

R

1

O

O H

R R

O

O C

R

1

+

O R

H

O HO

R1

R

1

O O O

R P

R

1

O

kcat/knon ≈ 103

Analogue stable de l’état de transition “phosphonate”

5b. Anticorps catalytiques Les premiers anticorps monoclonaux catalytiques: réaction estérase

reaction esterases

kun

kcat

kcat/ kun

KTx

KI (hapten )

1.2 × 10–6

3.1 × 10–4

2.7 × 102

5.6 × 10-8

2.4 × 10-6

3.2 × 10–6

20

6.3 × 106

2.5 × 10-10

5.0 × 10-7

Origine du pouvoir catalytique des enzymes Acceleration des réactions par catalyse enzymatique (kcat/knon)

1. Catalyse générale acido-basique Hydrolyse non-catalysée d’un acétale: séparation de charges dans l’état de transition. Situation défavorable, réaction lente O

O OR

δ+

δ−

OR

Catalyse acide SPECIFIQUE: Fixation de H+ dans l’état de transition (pH !) Catalyse acide GENERALE: transfert de H+ dans l’état de transition. La neutralisation de la charge stabilise l’état de transition, accelération de la réaction O

O OR

H

A

δ+

OR H

A

δ−

1. Catalyse générale acido-basique Hydrolyse non-catalysée d’un ester : séparation de charges dans l’état de transition. Situation défavorable, réaction lente O R'

C

O δ− OR

R'

C

+ O H

O H

H

OR

δ+

H

O R'

δ−

C

O

OR

R'

C

+ O H

O H

H

OR

δ−

H

δ+ δ+

NH2 NH2 B

B

1. Catalyse générale acido-basique

Catalyse dénomée “générale” pour la distinuger de la catalyse “spécifique” (H+ ou OH-) Si la catalyse acido-basique a lieu en même temps: catalyse concertée

1. Catalyse générale acido-basique

1. Catalyse générale acido-basique: réaction Rnase A

RNase A

3. Catalyse par des ions métaliques

Deux classes d’enzymes: métalloenzymes (Fe2+, Fe3+, Cu2+, Zn2+, Mg2+, Mn2+, ou Co3+) enzymes activées par des ions métaliques (Na+, K+, Mg2+, or Ca2+) Participent aux réactions: Fixation des substrats Réaction d’oxydo-réduction Stabilisation électrostatique

4. Catalyse covalente

Pyridoxal phosphate H

O C

H

O O O

P

O

O N

CH3

H

coenzyme PLP R

R

NH2

H H C

O P

O

H

O

H

O O

N C

O

Schiff Base

O O

P

O

O N

O N H

CH3

H

CH3

4. Catalyse covalente Exemple: l’Arginine Decarboxylase An extremely unstable intermediate

Spontaneous reaction O

O-

H

C

(-)C

CH R

H R

HC

+ CO2

NH2

NH2 H

NH2

R

-17 s-1 kuncatalyzed: ~ 2 x 10–17 –1

k = 2 × 10

s

-OH

HO

NH H2 C

R=

H2 C

H2 C

H N

C

Arginine decarboxylase + Enz-PLP

-O

O-

O C HC

O

R

NH2

NH2

A stable intermediate BH

H

C

C

CH R

N

H R

+ CO2

HC

+ Enz-PLP R

NH2

+HN

O-

2-O PO 3

O-

2-O PO 3

N H N H+

PLP alone: k = 3.9 × 10–6 s–1 Arg decarboxylase: = 1375 kcat =kcat 1375 s-1 s–1 kPLP/knon = 2 × 1011 kcat/knon = 7 × 1019

5. Stabilization de l’état de transition

5. Stabilization de l’état de transition

1. Catalyse générale acido-basique: réaction catalysée par l’Enolase general base catalyst Lys 3 4 5

HOOC-Glu 2 1 1

general acid catalyst

H

H2 O

Lys 345

H

H2 N:

-

OH

O

O

P

H O O-

O

O

+ O

H2 C

H2 NH+

-

O

-

O O-

2-phosphoglycerate

OOC-Glu 211

P O

O

-

-

phosphoenolpyruvate

HOOC-Glu 211

Exemple optionnel

Lys 345

H H

+

OH

H2 NH

O -

O O-

O O

P O

-

-

Intermédiaire ene-diolate

3. Entropy: entropy loss in the formation of EA The rotational and translational entropies of the substrate have been lost already during formation of EA complex

example: Strain/distortion

Transition state: Enzyme stablize T.S. to accelerate the reaction rate. Enzyme should bind tighter in T.S. than in substrate and product states.

example: Proline racemase and Isocitrate lyase (Prof. Robert Abeles)

Low barrier hydrogen bonds and enzymatic catalysis