DRUG TARGETS: ENZYMES

Chapter 3

Naming enzymes

• Root + ase

• Classification Reaction Type CatalyzedOxidoreductases Oxidation Reduction Reactions

Transferases Group transfer Reactions

Hydrolases Hydrolysis Reactions

Lyases Addition or removal of groups to form

double bonds

Isomerases Intramolecular group transfers

Ligases Joining two substrates

Structure and function of enzymes

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

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 reaction

Structure and function of enzymes

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 DG remains the same

Methods of enzyme catalysis

• Provides a reaction surface (the active site)

• Provides a suitable environment (hydrophobic)

• Brings reactants together

• Positions reactants correctly for reaction

• Weakens bonds in the reactants

• Provides acid / base catalysis

• Provides nucleophilic groups

Structure and function of enzymes

The active site

• Hydrophobic hollow or cleft on the enzyme surface

• Accepts reactants (substrates and cofactors)

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

ENZYME

Active siteActive site

Substrate binding

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

groups in the substrate and functional groups in the active site

Induced fit

Induced fit

SubstrateS

Substrate binding

• Ionic• H-bonding• van der Waals

Bonding forces

Example

S

Enzyme

Active site

vdwinteraction

ionicbond

H-bond

PheSer

OH

Asp

CO2

H3CC

C

O

O

O

Substrate binding

• Ionic• H-bonding• van der Waals

Bonding forces

Example - Binding of pyruvic acid in LDH

H-Bond

Possible interactions vdw-interactions

O

H

H3N

H3CC

C

O

O

OIonic bond

H-Bond

H3CC

C

O

O

O

van der Waals

H3CC

C

O

O

O

Ionic

H3CC

C

O

O

O

Substrate binding

• Induced fit - Active site alters shape to maximise intermolecularbonding

Bonding forces

Intermolecular bonds not optimum length for maximum bonding

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

S Phe

SerO

H

Asp

CO2 Induced fit

SPhe

Ser

OH

Asp

CO2

Substrate binding

Example - Binding of pyruvic acid in LDH

O

H

H3N

H3CC

C

O

O

O

O

O

O

Substrate binding

Example - Binding of pyruvic acid in LDH

O

H

H3N

p bondweakened

H3CC

C

O

O

O

Catalysis mechanisms

• Histidine

Acid/base catalysis

Nucleophilic residues

NNH

+H

-H NNH

H

Non-ionizedActs as a basic catalyst(proton 'sink')

IonizedActs as an acid catalyst(proton source)

H3N CO2

OH

H

L-Serine

H3N CO2

SH

H

L-Cysteine

Catalysis mechanisms

OH

Ser

X

Substrate

O

Ser

H2O

OH

Ser

HOProduct

Serine acting as a nucleophile

Catalysis mechanisms

Mechanism for chymotrypsin to hydrolyses peptide bonds

Catalytic triad of serine, histidine and aspartate

Chymotrypsin

NNO H

H

O O

Ser His Asp

..:

:

Catalysis mechanisms

Mechanism for chymotrypsin

ProteinNH

C

O

Protein

: :

Chymotrypsin

NNO H

H

O O

Ser His Asp

..:

:

Catalysis mechanisms

Mechanism for chymotrypsin

O

ProteinNHC

O

Protein

: :

:Chymotrypsin

NNH

H

O O

Ser His Asp

::

Catalysis mechanisms

Mechanism for chymotrypsin

NO

ProteinNHC

O

Protein

: :

:

:H

Chymotrypsin

NH

O O

Ser His Asp

: :

Catalysis mechanisms

Mechanism for chymotrypsin

O

C

:

OProtein

: :

HO

H

: :

Chymotrypsin

NNH

O O

Ser His Asp

::

:

Catalysis mechanisms

Mechanism for chymotrypsin

O

HO

C

O

Protein

: :

:

H

Chymotrypsin

NNH

O O

Ser His Asp

::

:

Catalysis mechanisms

Mechanism for chymotrypsin

:N

O

OC

O

Protein

: :

:

H

::

H

Chymotrypsin

NH

O O

Ser His Asp

: :

Catalysis mechanisms

Mechanism for chymotrypsin

OH

C

O

Protein

: :

Chymotrypsin

NNO H

H

O O

Ser His Asp

..:

:

Catalysis mechanisms

Mechanism for chymotrypsin

NNOH

Cprotein

HO O

Ser His Asp

OH

O

: :..

NNO H

proteinNHC

O

protein

HO O

Ser His Asp

:

..:

:

:

NNO H

proteinNHC

O

protein

HO O

Ser His Asp

:

:

:

:

..

NNO

proteinNHC

O

protein

HO O

Ser His Asp

H

:

:

:..

: NNO

CO

HO O

Ser His Asp

:

: : :

:proteinO

H H

: :

NNO

C

O

protein

HO O

Ser His Asp

O

H

H

: ::

: :..

..

NNO

C

O

protein

HO O

Ser His Asp

OH

H: :

: :..

..

..

Overall process of enzyme catalysis

S

E

ES

P

E

EP

P

E

E + P

E

S

E + S

E

• Binding interactions must be strong enough to hold the substrate sufficiently long for the reaction to occur

• Interactions must be weak enough to allow the product to depart • Implies a fine balance• Designing molecules with stronger binding interactions results in

enzyme inhibitors which block the active site

Regulation of enzymes• Many enzymes are regulated by agents within the cell • Regulation may enhance or inhibit the enzyme • The products of some enzymes may act as inhibitors • Usually bind to a binding site called an allosteric binding site

Example

Phosphorylase a

Glucose-1-phosphate

HO

O

H

H

HO

H

O

OHHH

OH

P

OH

O

HO

OO

H

H

HO

H

OHOHH

H

OH

Glycogen

n

N

NN

N

NH2

O

OHOH

HH

HH

OPO

O

O

AMP

ACTIVE SITE (open)

ENZYMEEnzyme

Regulation of enzymes

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

Allostericbinding site

Active site

(open)ENZYMEEnzyme

Inducedfit

Active siteunrecognisable

Allostericinhibitor

Regulation of enzymes

• Enzymes with allosteric sites are often at the start of a biosynthetic pathway

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

P’’’P’’P’

Biosynthetic pathway

Feedback controlInhibition

PS

(open)ENZYMEEnzyme

Proteinkinase

Cell

Regulation of enzymes• External signals can regulate the activity of enzymes

(e.g. neurotransmitters or hormones)• Chemical messenger initiates a signal cascade which activates

enzymes called protein kinases• Protein kinases phosphorylate target enzymes to affect activity

Example

Adrenaline

Signal cascade

Phosphorylase b (inactive)

Phosphorylase a (active)

Glycogen Glucose-1-phosphate