topic 3 proteins as drug targets - university of wisconsin-eau

Topic 3 Proteins as DrugTargets

Enzymes-Chapter 3, 4 Patrick andPart I Corey

Enzymes as TargetsEnzymes as Targets

Structure and function of enzymesThe active siteSubstrate binding

Induced fitBonding forces

Catalysis mechanismsAcid/base catalysisNucleophilic residues

Overall process of enzyme catalysisCompetitive (reversible) inhibitorsNon competitive (irreversible) inhibitorsNon competitive (reversible) allosteric inhibitors

Example: NSAIDS for inflammation

Structure and function of enzymesStructure and function of enzymes

•• Globular proteins acting as the bodyGlobular proteins acting as the body’’s catalystss catalysts•• Speed up time for reaction to reach equilibriumSpeed up time for reaction to reach equilibrium•• Lower the activation energy of a reactionLower the activation energy of a reaction

Example:Example:

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

LDH

Pyruvic acid Lactic acid

H3C

C

C

O

OHO

C O

CH3C

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 ΔΔGGremains the sameremains 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 reactants (transition state)Weaken bonds in the reactants (transition state)

•• Provide acid / base catalysisProvide acid / base catalysis

•• Provide Provide nucleophilesnucleophiles

1Structure and function of enzymes1Structure and function of enzymes

The active siteThe 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

Substrate bindingSubstrate 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 functionalBinding 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

Induced fitInduced fit

Induced fitInduced fit

SubstrateSubstrateSS

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

Bonding forcesBonding forces

Example:Example:

SS

Enzyme

Active site

vdwvdwinteractioninteraction

ionicionicbondbond

H-bondH-bond

PheSer

OH

Asp

CO2


van der WaalsH-Bond

Ionic

H3C

C

C

O

O

O

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


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

OH

H3N

H-Bond

Ionicbond

Possible interactions vdw-interactions

H3C

C

C

O

O

O


•• Induced fit - Active site alters shape to maximiseInduced fit - Active site alters shape to maximiseintermolecular bondingintermolecular bonding


Intermolecular bonds notoptimum length formaximum bonding

Intermolecular bond lengths optimisedSusceptible bonds in substrate strainedSusceptible bonds in substrate moreeasily broken

SS Phe

SerO H

Asp

CO2 Inducedfit

SSPhe

SerO H

Asp

CO2



O

H

H3N

H3C

C

C

O

O

O

O

O

O



O

H

H3N

pi bondpi bondweakenedweakened

H3C

C

C

O

O

O


Catalysis mechanismsCatalysis mechanisms

•• HistidineHistidine

4.1 Acid/base catalysis4.1 Acid/base catalysis

4.2 Nucleophilic residues4.2 Nucleophilic residues

N

NH

+H

-H N

NH

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

HOProduct

Serine acting as a Serine acting as a nucleophilenucleophile

Catalysis mechanismsCatalysis mechanisms

Overall process of enzyme catalysisOverall 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 bindingDrug design - designing molecules with stronger binding

interactions results in enzyme inhibitors which block the activeinteractions results in enzyme inhibitors which block the activesitesite

Competitive (reversible) inhibitorsCompetitive (reversible) inhibitors

•• Inhibitor binds reversibly to the active siteInhibitor 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 andInhibition depends on the strength of inhibitor binding and

inhibitor concentrationinhibitor concentration•• Substrate is blocked from the active siteSubstrate 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

Non competitive (irreversible) inhibitorsNon 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

Non competitive (irreversible) inhibitorsNon competitive (irreversible) inhibitorsX

OH OH

X

O

Covalent Bond

Irreversible inhibitionExample: aspirin for COX (PGH2 synthase)

ACTIVE SITE (open)

ENZYMEEnzyme

Non competitive (reversible) Non competitive (reversible) allosteric allosteric inhibitorsinhibitors

•• 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

Non competitive (reversible) Non competitive (reversible) allosteric allosteric inhibitorsinhibitors

•• Enzymes with allosteric sites often at start of biosyntheticEnzymes with allosteric sites often at start of biosyntheticpathwayspathways

•• 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 offFinal 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

EXAMPLE: NSAIDS for inflammation

topic 3 proteins as drug targets - university of wisconsin-eau

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