Patrick Patrick An Introduction to Medicinal An Introduction to Medicinal

ChemistryChemistry 3/e 3/e

Chapter 4Chapter 4

PROTEINS AS DRUG PROTEINS AS DRUG TARGETS:TARGETS:ENZYMESENZYMES

ContentsContents

1. Structure and function of enzymes (3 slides)2. The active site3. Substrate binding

3.1. Induced fit3.2. Bonding forces (5 slides)

4. Catalysis mechanisms4.1. Acid/base catalysis4.2. Nucleophilic residues

5. Overall process of enzyme catalysis6. Competitive (reversible) inhibitors7. Non competitive (irreversible) inhibitors8. Non competitive (reversible) allosteric inhibitors (2 slides)

[18 slides]

1. Structure and function of enzymes1. Structure and function of enzymes

• Globular proteins acting as the body’s Globular proteins acting as the body’s catalystscatalysts

• Speed up time for reaction to reach Speed up time for reaction to reach equilibriumequilibrium

• Lower the activation energy of a reaction by Lower the activation energy of a reaction by stabilizing the transition statestabilizing the transition stateExample:Example:

LDH = Lactate dehydrogenase (enzyme)NADH2 = Nicotinamide adenosine dinucleotide (reducing agent & cofactor)Pyruvic acid = Substrate

LDH

Pyruvic acid Lactic acid

H3CC

CO

O HO

C OCH3C

H

NADH2 NAD+++

OH OH

Lowering the activation energy of reactionLowering the activation energy of reaction

Act. energy

Transition state

WITHOUT ENZYME

Product

Startingmaterial

Energy

WITH ENZYME

Product

Startingmaterial

Energy

∆G

Newtransition

state

∆G

Act. energy

• Enzymes lower the activation energy of a reaction but Enzymes lower the activation energy of a reaction but G G remains the same.remains the same.

1. Structure and function of enzymes1. Structure and function of enzymes

Methods of enzyme catalysisMethods of enzyme catalysis

• Provide a reaction surface (the active site)Provide a reaction surface (the active site)

• Provide a suitable environment (hydrophobic)Provide a suitable environment (hydrophobic)

• Bring reactants togetherBring reactants together

• Position reactants correctly for reactionPosition reactants correctly for reaction

• Weaken bonds in the reactantsWeaken bonds in the reactants

• Provide acid / base catalysisProvide acid / base catalysis

• Provide nucleophilesProvide nucleophiles

1. Structure and function of enzymes1. Structure and function of enzymes

2. The active site2. The active site

• Hydrophobic hollow or cleft on the enzyme surfaceHydrophobic hollow or cleft on the enzyme surface

• Accepts reactants (substrates and cofactors)Accepts reactants (substrates and cofactors)

• Contains amino acids whichContains amino acids which - bind reactants (substrates and cofactors)- bind reactants (substrates and cofactors) - catalyse the reaction- catalyse the reaction

ENZYME

Active siteActive site

3. Substrate binding3. Substrate binding

• Active site is nearly the correct shape for the substrateActive site is nearly the correct shape for the substrate• Binding alters the shape of the enzyme (induced fit)Binding alters the shape of the enzyme (induced fit)• Binding will strain bonds in the substrateBinding will strain bonds in the substrate• Binding involves intermolecular bonds between functional Binding involves intermolecular bonds between functional

groups in the substrate and functional groups in the active sitegroups in the substrate and functional groups in the active site

3.1 Induced fit3.1 Induced fit

Induced fitInduced fit

SubstrateSubstrateSS

• IonicIonic• H-bondingH-bonding• van der Waalsvan der Waals

3.2 Bonding forces3.2 Bonding forces

Example:Example:

SS

Enzyme

Active site

vdwvdwinteractioninteraction

ionicionicbondbond

H-bondH-bond

PheSer

OH

Asp

CO2

3. Substrate binding3. Substrate binding

van der WaalsH-Bond

Ionic

H3CC

C

O

O

O

• IonicIonic• H-bondingH-bonding• van der Waalsvan der Waals

3.2 Bonding forces3.2 Bonding forces

Example:Example: Binding of pyruvic acid in LDH Binding of pyruvic acid in LDH

O

H

H3N

H-Bond

Ionicbond

Possible interactions vdw-interactions

H3CC

C

O

O

O

3. Substrate binding3. Substrate binding

• Induced fit - Active site alters shape to maximise Induced fit - Active site alters shape to maximise intermolecular bondingintermolecular bonding

3.2 Bonding forces3.2 Bonding forces

Intermolecular bonds not optimum length for maximum bonding

Intermolecular bond lengths optimisedSusceptible bonds in substrate strainedSusceptible bonds in substrate more easily broken

SS Phe

SerO

H

Asp

CO2 Induced fit

SSPhe

Ser

OH

Asp

CO2

3. Substrate binding3. Substrate binding

Example:Example: Binding of pyruvic acid in LDH Binding of pyruvic acid in LDH

O

H

H3N

H3CC

C

O

O

O

O

O

O

3. Substrate binding3. Substrate binding

Example:Example: Binding of pyruvic acid in LDH Binding of pyruvic acid in LDH

O

H

H3N

pi bondpi bondweakenedweakened

H3CC

C

O

O

O

3. Substrate binding3. Substrate binding

4. Catalysis mechanisms4. Catalysis mechanisms

• HistidineHistidine

4.1 Acid/base catalysis4.1 Acid/base catalysis

4.2 Nucleophilic residues4.2 Nucleophilic residues

NNH

+H

-H NNH

H

Non-ionisedNon-ionisedActs as a basic catalystActs as a basic catalyst(proton 'sink')(proton 'sink')

IonisedIonisedActs as an acid catalystActs as an acid catalyst(proton source)(proton source)

H3N CO2

OH

H

L-SerineL-Serine

H3N CO2

SH

H

L-CysteineL-Cysteine

OH

Ser

X

Substrate

O

Ser

H2O

OH

Ser

HO Product

Serine acting as a nucleophileSerine acting as a nucleophile

4. Catalysis mechanisms4. Catalysis mechanisms

5. Overall process of enzyme catalysis5. Overall process of enzyme catalysis

SS

EE

ES

PP

EE

EP

PP

EE

E + P

EE

SS

E + S

EE

• Binding interactions must be;Binding interactions must be; - strong enough to hold the substrate sufficiently long for the - strong enough to hold the substrate sufficiently long for the

reaction to occurreaction to occur - weak enough to allow the product to depart - weak enough to allow the product to depart • Implies a fine balanceImplies a fine balance• Drug design - designing molecules with stronger binding Drug design - designing molecules with stronger binding

interactions results in enzyme inhibitors which block the active interactions results in enzyme inhibitors which block the active sitesite

6. Competitive (reversible) inhibitors6. Competitive (reversible) inhibitors

• Inhibitor binds reversibly to the active site Inhibitor binds reversibly to the active site • Intermolecular bonds are involved in bindingIntermolecular bonds are involved in binding• No reaction takes place on the inhibitorNo reaction takes place on the inhibitor• Inhibition depends on the strength of inhibitor binding and Inhibition depends on the strength of inhibitor binding and

inhibitor concentrationinhibitor concentration• Substrate is blocked from the active site Substrate is blocked from the active site • Increasing substrate concentration reverses inhibitionIncreasing substrate concentration reverses inhibition• Inhibitor likely to be similar in structure to the substrateInhibitor likely to be similar in structure to the substrate

II

EEEE

SS

II

EE

7. Non competitive (irreversible) inhibitors7. Non competitive (irreversible) inhibitors

• Inhibitor binds irreversibly to the active site Inhibitor binds irreversibly to the active site • Covalent bond formed between the drug and the enzymeCovalent bond formed between the drug and the enzyme• Substrate is blocked from the active site Substrate is blocked from the active site • Increasing substrate concentration does not reverse inhibitionIncreasing substrate concentration does not reverse inhibition• Inhibitor likely to be similar in structure to the substrateInhibitor likely to be similar in structure to the substrate

X

OH OH

X

O

Covalent Bond

Irreversible inhibition

ACTIVE SITE (open)

ENZYMEEnzyme

8. Non competitive (reversible) allosteric inhibitors8. Non competitive (reversible) allosteric inhibitors

• Inhibitor binds reversibly to the allosteric site Inhibitor binds reversibly to the allosteric site • Intermolecular bonds are formedIntermolecular bonds are formed• Induced fit alters the shape of the enzymeInduced fit alters the shape of the enzyme• Active site is distorted and is not recognised by the substrateActive site is distorted and is not recognised by the substrate• Increasing substrate concentration does not reverse inhibitionIncreasing substrate concentration does not reverse inhibition• Inhibitor is not similar in structure to the substrateInhibitor is not similar in structure to the substrate

AllostericAllostericsitesite

Active siteActive site

(open)ENZYMEEnzyme

Inducedfit

Active siteActive siteunrecognisableunrecognisable

Allostericinhibitor

8. Non competitive (reversible) allosteric inhibitors8. Non competitive (reversible) allosteric inhibitors

• Enzymes with allosteric sites often at start of biosynthetic Enzymes with allosteric sites often at start of biosynthetic pathwayspathways

• Enzyme is controlled by the final product of the pathwayEnzyme is controlled by the final product of the pathway• Final product binds to the allosteric site and switches off Final product binds to the allosteric site and switches off

enzymeenzyme• Inhibitor may have a similar structure to the final productInhibitor may have a similar structure to the final product

P’’’P’’P’

Biosynthetic pathway

Feedback controlInhibition

PPSS

(open)ENZYMEEnzyme