Enzyme Kinetics & Inhibitors PDF

ENZYMES/DRUG TARGETS Dr. Ritesh Raju Department of Pharmacology © Oxford University Press, 2013 Learning Objectives 1. Describe enzymes as specialised proteins that catalyse biochemical reactions (catalytic mechanism of chymotrypsin) 2. Explain the kinetics of enzyme reactions and the modes of enzym...

ENZYMES/DRUG TARGETS Dr. Ritesh Raju Department of Pharmacology © Oxford University Press, 2013 Learning Objectives 1. Describe enzymes as specialised proteins that catalyse biochemical reactions (catalytic mechanism of chymotrypsin) 2. Explain the kinetics of enzyme reactions and the modes of enzyme inhibition, both reversible and irreversible 3. Illustrate the role of enzymes in metabolism and disease and how they can therefore be used as biomarkers of injury to specific organs © Oxford University Press, 2013 1. Structure and function of enzymes Notes Globular proteins acting as the body’s catalysts Speed up time for reaction to reach equilibrium Lower the activation energy of a reaction Example: O NADH + H3C C Pyruvic acid O C OH LDH H HO H3C C Lactic acid O C + NAD+ OH LDH = Lactate dehydrogenase (enzyme) NADH = Nicotinamide adenosine dinucleotide (reducing agent & cofactor) Pyruvic acid = Substrate © Oxford University Press, 2013 1. Structure and function of enzymes Lowering the activation energy of reaction Energy Energy Transition state New transition state Act. energy Starting material Act. energy Starting material ∆G ∆G Product Product WITHOUT ENZYME WITH ENZYME Note Enzymes lower the activation energy of a reaction but DG remains the same © Oxford University Press, 2013 1. Structure and function of enzymes 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 Stabilises the transition state with intermolecular bonds © Oxford University Press, 2013 2. 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) - participate in the enzyme-catalysed reaction Active site Active site ENZYME © Oxford University Press, 2013 3. Substrate Binding 3.1 Induced fit Substrate S Induced fit Notes: Active site is nearly the correct shape for the substrate Binding alters the shape of the enzyme (induced fit) The binding process can strain bonds in the substrate Binding involves intermolecular bonds between functional groups in the substrate and functional groups in the active site © Oxford University Press, 2013 3. Substrate Binding 3.2 Bonding forces Ionic H-bonding van der Waals Example vdw interaction S H-bond Active site Ser O Ionic bond H Phe CO2 Asp Enzyme © Oxford University Press, 2013 3. Substrate Binding 3.2 Bonding forces Ionic H-bonding van der Waals Example - Binding of pyruvic acid in LDH O O H-Bond H3C H O C O C O Possible interactions O C H3C vdw-interactions C O + H3N Ionic bond H-Bond van der Waals Ionic © Oxford University Press, 2013 3. Substrate Binding 3.2 Bonding forces Induced fit - Active site alters shape to maximise intermolecular bonding S Ser O S Ser Phe H CO2 Asp Intermolecular bonds not optimum length for maximum bonding Induced fit O H Phe CO2 Asp Intermolecular bond lengths optimised Susceptible bonds in substrate strained Susceptible bonds in substrate more easily broken © Oxford University Press, 2013 3. Substrate Binding Example - Binding of pyruvic acid in LDH O H O O C H3C C O + H3N © Oxford University Press, 2013 3. Substrate Binding Example - Binding of pyruvic acid in LDH Induced Fit O p bond weakened H O O C H3C C + H3N O © Oxford University Press, 2013 4. Catalysis mechanisms 4.1 Acid/base catalysis Histidine +H+ NH N NH -H+ N H Non-ionised Acts as a basic catalyst (proton 'sink') Ionised Acts as an acid catalyst (proton source) Aspartic acid O O -H+ H O Non-ionised Acts as an acid catalyst (proton source) +H+ O Ionised Acts as a basic catalyst (proton ‘sink’) © Oxford University Press, 2013 4. Catalysis mechanisms 4.2 Nucleophilic residues H H H3N L-Serine CO2 OH H3N CO2 SH L-Cysteine © Oxford University Press, 2013 4. Catalysis mechanisms Serine acting as a nucleophile Substrate X HO OH Ser O Ser H2O Product OH Ser © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin Catalytic triad of serine, histidine and aspartate Serine acts as a nucleophile Histidine acts as an acid/base catalyst Aspartate orientates histidine.. :O Ser :N N H H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin : O: C Protein.. :O Ser NH Protein :N N H H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin : :O: Protein C :O Ser NH Protein :N N H H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin : :O: Protein C :O: Ser NH Protein H + N N H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin H H : : : Ser : O :N N H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin : :O: Protein O C :O: Ser H H :N N H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin : :O: H O: Protein : C H :O : : Ser N N H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin :O: C OH Protein.. :O Ser :N N H H O His O Asp Chymotrypsin © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin :O: Ser C C Ser His O Asp Ser.. H :O: C :O : Ser O.. His O O N H His O Asp O :O : Ser H O :N :O : Ser Asp protein.. : O :.. OH protein.. C H :N C N H N H O NH protein :O : O Asp protein H N H :N H protein O : NH protein O :.. :O : :O protein His.. :O : protein N H :N H :.. :O NH protein : C protein N H His C O O Asp OH O H N N H His O Asp O.. : OH Ser :N N H His O O Asp © Oxford University Press, 2013 4. Catalysis mechanisms Mechanism for chymotrypsin – the oxyanion hole Region of the active site occupied by the negatively charged oxygen of the tetrahedral intermediate Oxyanion stabilised by hydrogen bonds to peptide bonds close by Interaction stabilises transition state leading to the oxyanion intermediate and lowers the activation energy O O N N H H : :O: Protein C :O: Ser NH Protein H + N N H O His O Asp Chymotrypsin © Oxford University Press, 2013 5. Overall Process of Enzyme Catalysis S P S EE E+S E ES P E EP E E+P Notes: 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 Interactions stabilise the transition state Designing molecules with stronger binding interactions results in enzyme inhibitors which block the active site © Oxford University Press, 2013 6. Regulation of Enzymes Many enzymes are regulated by agents within the cell Regulation may enhance or inhibit the enzyme The products of some enzyme-catalysed reactions may act as inhibitors Usually bind to a binding site called an allosteric binding site NH2 Example N N O O P O O O H H OH H OH H O HO HO H H H AMP H O H O HO N H OH H OH Glycogen N OH OH Phosphorylase a H H H OH O Glucose-1-phosphate O P HO OH © Oxford University Press, 2013 n 6. Regulation of Enzymes Active site unrecognisable Active site ACTIVE SITE (open) Enzyme ENZYME Allosteric binding site Induced fit (open) Enzyme ENZYME Allosteric inhibitor 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 differs in structure to the substrate © Oxford University Press, 2013 6. Regulation of Enzymes Biosynthetic pathway S P P’ P’’ P’’’ (open) Enzyme ENZYME Inhibition Feedback control Notes Enzymes with allosteric sites are often at the start of a biosynthetic pathway The enzyme is controlled by the final product of the pathway The final product binds to the allosteric site and switches off the enzyme Drugs acting as enzyme inhibitors can be designed to target the active site or the allosteric site © Oxford University Press, 2013 6. 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 Phosphorylase b (inactive) Protein kinase Signal cascade Adrenaline Phosphorylase a (active) Glycogen Glucose-1-phosphate Cell © Oxford University Press, 2013 1. Overall Process of Enzyme Catalysis S P S EE E+S E ES P E EP E E+P Notes 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 © Oxford University Press, 2013 2. Reversible Inhibitors S I I EE E Notes Inhibitor binds reversibly to the active site Intermolecular bonds are involved in binding The inhibitor undergoes no reaction Inhibition depends on the strength of inhibitor binding and inhibitor concentration Substrate is blocked from the active site Increasing substrate concentration reverses inhibition Inhibitor likely to be similar in structure to substrate, product or cofactor © Oxford University Press, 2013 2. Reversible Inhibitors O N HS Examples O O N Me O N S N Me O ACE Inhibitors NH2 Me HO O Diuretics O O O O S N O Me H HN N H Statins HN O Cl N N O O O Me O N H Cl N H N F O NH O OH Sulphonamides N Me O O H2N O CO2H Me Protease inhibitors N Antidepressants Kinase inhibitors © Oxford University Press, 2013 3. Irreversible Inhibitors X Covalent Bond X OH OH O Irreversible inhibition Notes Inhibitor binds irreversibly to the active site Covalent bond formed between the drug and the enzyme Substrate is blocked from the active site Increasing substrate concentration does not reverse inhibition Inhibitor likely to be similar in structure to the substrate © Oxford University Press, 2013 3. Irreversible Inhibitors Examples Me H N O Me O P F O Me Nerve gases H S Me N N H3C Me O CH3 S H N Penicillins S O CH3 S H3C CO2H N S S Treatment of alcoholism H N O Me O Cephalosporins CO2H O H N O Me O Me Me O O N H N S O N Me Me O Proton pump inhibitors Me Me O O Anti-obesity H © Oxford University Press, 2013 Me 3. Irreversible Inhibitors Examples - orlistat Orlistat C6H13 C11H23 O O O C6H13 But H C11H23 O O NHCHO C11H23 O O O C6H13 Ser O Pancreatic lipase Ser But NHCHO Pancreatic lipase O But OH O O NHCHO O O Ser Pancreatic lipase Notes Orlistat is an anti-obesity drug that inhibits pancreatic lipase The enzyme is blocked from digesting fats in the intestine Fatty acids and glycerol are less absorbed as a result Leads to reduced biosynthesis of fat in the body © Oxford University Press, 2013 4. Allosteric Inhibitors Active site unrecognisable Active site ACTIVE SITE (open) Enzyme ENZYME Allosteric binding site Induced fit (open) Enzyme ENZYME Allosteric inhibitor Notes Inhibitor binds reversibly to the allosteric 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 © Oxford University Press, 2013 4. Allosteric Inhibitors Example: 6-Mercaptopurine SH N N N N H Notes Inhibits the first enzyme in the biosynthesis of purines Blocks the biosynthesis of purines and DNA Used in the treatment of leukaemia © Oxford University Press, 2013 5. Transition-state Inhibitors Notes Drugs designed to mimic the transition state of an enzyme-catalysed reaction Transition-state inhibitors are likely to bind more strongly than drugs mimicking the substrate or product Transition states are high energy, transient species and cannot be isolated or synthesised Drug design can be based on reaction intermediates which are closer in character to transition states than substrates or products Design a drug that mimics the stereochemistry and binding properties of the reaction intermediate, but is stable. © Oxford University Press, 2013 5. Transition-state Inhibitors Example: Renin inhibitors Inhibitor Angiotensinogen Renin Angiotensin converting enzyme (ACE) Angiotensin I Angiotensin II Notes Renin inhibitors block synthesis of angiotensin I and II Angiotensin II constricts blood vessels and raises blood pressure Renin inhibitors act as antihypertensives (lower blood pressure) © Oxford University Press, 2013 5. Transition-state Inhibitors Example: Renin inhibitors Reaction mechanism Tetrahedral intermediate R1 R1 H N Peptide Peptide R2 O H H O O Asp O Peptide H N O O H H H O Asp O Asp O O Peptide H2N Peptide R2 CO2H H O Asp Renin R1 + R2 H O Renin Peptide O Asp O O O Asp Renin Notes Two aspartyl residues involved in enzyme-catalysed reaction Tetrahedral intermediate involved © Oxford University Press, 2013 5. Transition-state Inhibitors Example: Renin inhibitors Aliskiren MeO MeO CHMe2 H N Protein O O H2N O N H OH CHMe2 HO NH2 Me Me Protein OH Reaction intermediate Hydroxyethylene transition-state mimic Notes Aliskiren contains a hydroxyethylene transition-state mimic Mimics the tetrahedral geometry of the reaction intermediate Mimics one of the hydroxyl groups (binding group) Stable - no leaving group present © Oxford University Press, 2013 5. Transition-state Inhibitors Other examples ACE inhibitors Statins CH3 H HO CO2H N H HO2C OH H N O CO2H F Protease inhibitors N H3C O S N N O N H H N CH3 O O O H N H H CONH2 H N OH N H H H © Oxford University Press, 2013 6. Suicide Substrates Notes Agents which are converted to irreversible inhibitors by the enzymecatalysed reaction React with the target enzyme once formed Cl Cl S O O OH Tienilic acid O Notes Marketed as a diuretic Withdrawn due to interaction with cytochrome P450 enzymes Agent acts as a suicide substrate on cytochrome P450 enzymes © Oxford University Press, 2013 6. Suicide Substrates Tienilic acid Cyt P450 Cl Cl SH O S OH Cyt P450 NADPH O2 S O O Cyt P450 +H H O Ar Ar S S O Cyt P450 O Ar S S O O H O Cyt P450 H Ar Ar S -H2O H H S OH Suicide substrate H Cyt P450 S O S O Cyt P450 O Cl Oxidation Tienilic acid Alkylation Cl Cyt P450 O S S O Enzyme alkylated and inhibited S O © Oxford University Press, 2013 7. Enzyme targets for useful medications 7.1 Antibacterial agents Dihydropteroate synthetase, transpeptidase 7.2 Antiviral agents HIV reverse transcriptase, HIV protease, viral DNA polymerase 7.3 Anti-inflammatory agents Cyclooxygenase 7.4 Cholesterol lowering agents HMG-CoA reductase 7.5 Antidepressants Monoamine oxidase 7.6 Anticancer agents Tyrosine kinase, dihydrofolate reductase, thymidylate synthase, aromatase etc © Oxford University Press, 2013 7. Enzyme targets for useful medications 7.7 Antihypertensive agents Renin, angiotensin converting enzyme 7.8 Treatment of male erectile dysfunction Phosphodiesterase 7.9 Anti-gout agents Xanthine oxidase 7.10 Anti-ulcer agents Proton pump 7.11 Alzheimers disease Cholinesterases 7.12 Diuretics Carbonic anhydrase © Oxford University Press, 2013