MCB 2000 CH9: Enzyme Mechanisms + Inhibitors

What are enzyme catalytic strategies?

What are the 4?

Enzyme catalytic strategies = specific enzymatic chemical + physical methods → ↓Ea + ↑Rate

• Covalent catalysis

• General acid-base

• Metal ion catalysis

• Approximation + Orientation

How many catalytic strategies do enzymes typically use?

Usually 2-3

What is covalent catalysis?

Covalent catalysis = residue nucleophilic side chain in active site → covalent intermediate → product

What is general acid-base?

What is the key amino acid involved in this? Why?

General acid-base = residue side chain → accepts/donates proton @ right step → stabilizes TS

• Histidine → imidazole ring → easily flip b/w protonated/deprotonated @ physiological pH

What is metal ion catalysis?

What are 3 common metal ion examples?

What are 3 functions?

Metal ion catalysis = Metal cations in enzyme active site → stabilize O^-, polarize H₂O, electrophile (wants e^-)

• Zn²⁺, Mg²⁺, Fe^2/3+

What is approximation + orientation?

Approximation + Orientation = bind 2 substrates next to e/o @ correct position → ↑Rate

What are the 2 steps of covalent catalysis?

Why does this help lower activation energy?

What 3 amino acids have good nucleophilic side chains? Why?

1. Nucleophilic side chain in active site attacks substrate

   1. Covalent E-S intermediate

2. Intermediate = hydrolyzed → Product + Enzyme = released

2 smaller steps replace 1 big step (↓E_A barrier)

• Serine → Serine proteases = Chymotrypsin, Trypsin, Elastase

  • R = -CH₂-OH

• Cysteine → Proteases = Papain + Caspases

  • R = -CH₂-SH

    • S = more nucleophilic than O

• Lysine → Lyase → Aldolase

  • R = -CH₂-CH₂-CH₂-CH₂-NH₂

    • Amine forms Schiff Base w/ carbonyls

      • Schiff Base = temporary C=N

“Sad, Cry, Laugh”

What is the workhouse of acid-base catalysis? Why?

What environment influences equilibrium of deprotonation vs. protonation?

What term describes unique property of histidine?

What are 2 amino acids that act as bases or proton acceptors?

What is 1 amino acid that act as acids or proton donors?

Histidine = physiological pH = 7.4 ~ imidazole ring pKa = 6 → easily protonates/deprotonates

Local environment of active site

• Nearby (+) charge → stabilizes (-) charge

• Nearby (-) charge → stabilizes (+) charge

Amphoteric = acts as both acid + base

Aspartate + Glutamate → Carboxylates accept H⁺

Lysine → Amine donates H⁺

What fraction of enzymes have a metal cofactor?

What is enzyme is zinc involved in?

What do metal cofactors in active site do to water? How does it help the enzyme mentioned above?

• What acts as the nucleophile?

1/3 enzymes → metal cofactor

Carbonic anhydrase = CO₂ + H₂O ⇔ HCO₃^- (bicarbonate)

• Polarizes H₂O → easier to deprotonate → OH^- → attack CO₂

  • OH^- = Nucleophile

What enzyme is iron involved in?

What enzyme is magnesium involved in?

ATP can act as substrate w/o magnesium cofactor: (True/False)

Fe^2/3+ of cytochrome P450 = electrophile

• Fe^2/3+ attacks substrate w/ reactive O → heme group inside enzyme → breaks down drugs/toxins + produce hormones

Mg²⁺ of Kinase → bind + stabilize phosphate -PO₄^3- (VERY NEGATIVE) of ATP

• Mg²⁺ hold in correct geometry → stabilize (-) charge → phosphorylate target

FALSE → ATP requires Mg²⁺ cofactor

What are 3 requirements of approximation + orientation?

What is the issue w/ first 2 requirements in dilute solutions?

What is the solution? Give an example of an enzyme and its 2 substrates.

About what factor does approximation + orientation increase the reaction rate by?

POE

• Proximity = close together

• Orientation = correct geometry

• Energy = enough energy to react

Problem = substrates diffuse around, rarely make contact, wrong orientation

Solution = enzyme = 2 binding pockets → hold substrates close tg @ correct orientation

• ↑Local [A]:[B]

• Hexokinase → Glucose + ATP

  • Glycolysis Initiation = Glucose → G6P

↑Rate by 10⁵

What are the 2 competing effects of increasing temperature/heat on enzymes?

What three interactions does too much heat disrupt?

• Heat → ↑Speed of molecules

• Heat → ↓ Rigidity → Denaturation (10-20°C > optimum)

• H-bonds

• Hydrophobic interactions

• Salt bridges

What is optimum temperature for humans? What does it mean?

What is the high fever temperature for humans?

• What types of enzymes lose activity?

Optimum temperature = 37°C → Rate gain = Geometric loosening

High fever temperature = 41°C → DANGEROUS

• Temperature-Sensitive Enzymes

All enzymes have the same optimum temperature: (True/False)

What types of extremophile thrives in hot springs? Why are they able to do this?

FALSE → different enzymes = different optimum temperatures

• Thermophiles = bacterium/archaeon

  • Proteins = stable to 80°C+

    • ↑ H-bonds

    • ↑ Salt Bridges

    • ↑ Packing Tightness

What are the 4 major components of the Activity vs. Temperature curve?

1. ↑Temperature → ↑KE = move faster, more collisions, more energetic

   1. Clear E_A barrier @ faster rate

2. Optimum temperature = 37°

3. Loss of optimal conformation = active site = less rigid

4. High Heat → Denaturation

Why does pH affect pepsin and chymotrypsin activities differently?

What are their optimal pH’s?

What are the pKa’s?

What organ does each enzyme occupy?

Different residues → affected differently by conditions

Pepsin → pH = 2 → stomach

• 2 Aspartate = carboxylate pKa = 3.5

  • 1 protonated, other deprotonated

    • Only occurs @ pH = 8

Chymotrypsin → pH = 8 → small intestine

• Histidine = imidazole pKa = 6

  • MUST be deprotonated to deprotonate Ser195 → pH = 8

What type of forces are involved in reversible inhibitors? Give 3 examples.

What bond is NOT involved in reversible inhibition?

(Most/Some) drugs are reversible inhibitors.

• Give 3 examples + describe binding + what it does to body

Non-covalent forces

• H-bonds

• Ionic Interactions

• Hydrophobic packing

Covalent bonds ≠ involved in reversible inhibition

MOST = bind → slow enzyme → let go (allows body to clear drug)

• ACE inhibitors → blood pressure

• COX inhibitors → ibuprofen

• Statin → cholesterol

What do irreversible inhibitors do?

What must the cell do if an irreversible inhibitor binds to an enzyme?

What are 3 examples of irreversible inhibitors?

Irreversible inhibitors = form covalent bond w/ critical residue → Enzyme = dead

Cell MUST make new enzyme

• Penicillin

• Aspirin

• Nerve agents

What are the 3 types of reversible inhibition?

1. Competitive inhibition

2. Uncompetitive inhibition

3. Noncompetitive (allosteric) inhibition

What is competitive inhibition?

What does the reaction look like?

What is an example?

What are the kinetic effects on Vmax and Km? Why?

Competitive inhibitor = shaped like substrate → binds to active site

E + I ⇔ EI (dead end)

Malonate = looks like succinate → blocks succinate hydrogenase in TCA Cycle

• Vmax = unchanged

  • ↑[S] = can outcompete competitive inhibitor

• Km = increases

  • ↑[S] required to reach ½ Vmax → ↓Binding Affinity

What is uncompetitive inhibition?

• Can it bind free enzyme? Why or why not?

What does the reaction look like?

What are the kinetic effects on Vmax and Km? Why?

Uncompetitive inhibitor = binds to ES-complex → ESI complex

• CAN NOT bind to free enzyme

• Inhibitor binding site = forms AFTER ES-complex formed

ES + I ⇔ ESI

• Vmax = decreases

  • ↑[S] → ↑ES-Complex → ↑ESI Complex → ↑Inhibition

    • CANNOT outcompete uncompetitive inhibitor

• Km = decreases

  • Less free ES complexes in solution

  • ↓[S] required to reach ½ Vmax → ↑Binding Affinity

What is noncompetitive inhibition?

• Affected/Unaffected by presence/absence of substrate?

What does the reaction look like?

What are the kinetic effects on Vmax and Km? Why?

Noncompetitive inhibitor = binds to allosteric site

• Unaffected by presence/absence of substrate

E + I ⇔ EI

ES + I ⇔ ESI

• Vmax = decreases

  • Reduced [functional enzyme] → Inhibited

• Km = unchanged

  • ~[S] required to reach ½ Vmax → ~Binding Affinity

What type of inhibition is this graph? Why?

Competitive inhibition

• Vmax = same

• Km = increased

What type of inhibition is this graph? Why?

Uncompetitive inhibition

• Vmax = decreased

• Km = decreased

What type of inhibition is this graph? Why?

Noncompetitive (allosteric) Inhibition

• Vmax = decreased

• Km = same

What type of reversible inhibitor is methotrexate?

What type of reversible inhibitor is lithium?

What type of reversible inhibitor is ketamine?

Methotrexate = competitive inhibitor of folate

• Dihydrofolate reductase = thymidine production

• Chemotherapy, rheumatoid arthritis

Lithium = uncompetitive inhibitor

• Inositol monophosphatase = neuron signaling

• Bipolar disorder

Ketamine = noncompetitive inhibitor of glutamate

• NMDA receptor (channel) = receptor

• Anesthetic, Treatment-resistant depression

• Can be long-lasting after signal clears

What is a Lineweaver-Burk plot?

What is the y-axis and what is the x-axis?

What is the y-intercept and the x-intercept?

What is the slope?

Lineweaver-Burk plot = graphical double-reciprocal representation of Michaelis-Menten equation in enzyme kinetics

Y-axis = 1/v; X-axis = 1/[S]

Y-intercept = 1/Vmax; X-intercept = -1/Km

Slope = Km/Vmax (double reciprocal)

What happens to the x-intercept, y-intercepts, and slope of Lineweaver-Burk plot in Competitive Inhibition? (How does it relate to Km and Vmax)?

What is the key characteristic of Competitive Inhibitor Lineweaver-Burk plot?

Competitive Inhibition → Vmax = same; Km = increases

• X-Intercept = moves right (Km increases)

• Y-Intercept = same (Vmax same)

• Slope = increases (line = steeper)

Same y-intercept

What happens to the x-intercept, y-intercepts, and slope of Lineweaver-Burk plot in Uncompetitive Inhibition? (How does it relate to Km and Vmax)?

What is the key characteristic of Uncompetitive Inhibitor Lineweaver-Burk plot?

Uncompetitive Inhibition→ Vmax = decreases; Km = decreases

• X-Intercept = moves left (Km decreases → -1/Km = more negative)

• Y-Intercept = moves up (Vmax decreases → 1/Vmax = more positive)

• Slope = same (Vmax & Km Δby same factor)

Parallel lines (same slope)

What happens to the x-intercept, y-intercepts, and slope of Lineweaver-Burk plot in Noncompetitive Inhibition? (How does it relate to Km and Vmax)?

What is the key characteristic of Noncompetitive Inhibitor Lineweaver-Burk plot?

Noncompetitive Inhibition→ Vmax = decreases Km = same

• X-Intercept = same (Km same)

• Y-Intercept = moves up (Vmax decreases) → 1/Vmax = more positive)

• Slope = increases (line = steeper)

Same x-intercept (same Km)

What is irreversible inhibition?

What are 3 important examples?

What must happen for enzyme function to return?

Irreversible inhibition = permanent enzyme inhibition through covalent bond formation (b/w inhibitor + active site side-chain)

• Aspirin

• Penicillin

• Proton pump inhibitors

Cell MUST make new enzyme → function return

What are the 3 common active site residues that irreversible inhibitors form covalent bonds w/? Why?

“Smile, Cry, Happy”

• Serine

• Cysteine

• Histidine

Side chains = STRONG biological nucleophiles

What happens to the x-intercept, y-intercepts, and slope of Lineweaver-Burk plot in Noncompetitive Inhibition? (How does it relate to Km and Vmax)?

What is the key characteristic of Noncompetitive Inhibitor Lineweaver-Burk plot?

What type of reversible inhibition does this look like? How can the difference be determined?

Irreversible Inhibition→ Vmax = decreases Km = same

• X-Intercept = same (Km same)

• Y-Intercept = moves up (Vmax decreases) → 1/Vmax = more positive)

• Slope = increases (line = steeper)

Same x-intercept (same Km)

Looks like Noncompetitive Inhibition

• Differentiate → Time-Course Experiment

  • Loss of activity over time = irreversible inhibition

What are the 4 types of irreversible inhibitors? Describe the specificity of each type.

• Group-specific = chemical molecules = react w/ specific amino acid side chains of enzyme

  • Least specific (Ex. any serine on enzyme)

• Affinity label = chemical molecules = mimic substrate → react w/ enzyme active site

  • Reactive BEFORE binding

  • Only active site

• Mechanism-based = chemical molecules = mimic substrate → react w/ enzyme active site

  • Inert = reactive AFTER binding

    • Trick enzyme → normal catalysis → TRAPS + inactivates enzyme

  • Only active site

  • “Suicide Inhibitor”

• Transition-state analog = chemical molecules = mimic transition state of substrate → react w/ enzyme active site

  • DO NOT form covalent bonds

  • Only active site

What are the 4 types of irreversible inhibitors? Give an example of each.

• Group-specific = chemical molecules = react w/ specific amino acid side chains of enzyme

  • DIPF = reacts w/ every serine = useful for research = find catalytic Serines of enzymes

• Affinity label = chemical molecules = mimic substrate → react w/ enzyme active site

  • TPCK = substrate-shaped → tags His-57 of Chymotrypsin

• Mechanism-based = chemical molecules = mimic substrate → react w/ enzyme active site

  • Aspirin = blocks COX Enzymes

  • Penicillin = blocks Transpeptidase

• Transition-state analog = chemical molecules = mimic transition state of substrate → react w/ enzyme active site

  • Statin = blocks HMG-CoA reductase

Describe the mechanism of aspirin as a mechanism-based inhibitor of Cyclooxygenase (COX Enzyme).

• What are the reactants and products?

• What is the nucleophile and leaving group?

COX’s Ser530 = Nu → attacks aspirin’s acetyl carbonyl

• COX Ser530 + Aspirin → Salicylate + Acetylated COX

• Salicylate = LG = painkiller

• Acetylated COX= permanently acetylated = DEAD

What is the name of acid of aspirin?

Through what type of bond is acetyl group attached to aspirin by?

Why does aspirin have short-term effect?

What does low-dose daily aspirin help with? Why?

Aspirin = acetylsalicylic acid

Ester bond

Aspirin = short-term effect → most cells synthesize new COX Enzyme

Low-Dose Daily Aspirin → Platelets = cell fragments = NO nucleus → CANNOT synthesize new COX Enzyme (10 days)

• ↓Risk of heart attack + stroke

Describe the mechanism of penicillin as a mechanism-based inhibitor of Transpeptidase (Bacterial enzyme).

• What are the reactants and products?

• What is the nucleophile?

Transpeptidase Ser = Nu → attacks Penicillin β-Lactam Carbonyl

• Penicillin β-Lactam (4-membered) Ring = STRAIN = substrate-shaped

• Transpeptidase Ser + Penicillin → Transpeptidase Ser-Penicillin (Ring = open)

• Acetylated Transpeptidase = permanently acetylated = DEAD

What is the normal function of transpeptidase?

• What is peptidoglycan?

• What is the normal mechanism of transpeptidase?

What function is inhibited by Penicillin?

Why does penicillin not kill humans?

Transpeptidase = cross-link peptide chains in bacterial cell wall

• Peptidoglycan = sugar meshwork cross-linked by short peptide bonds = bacterial cell wall

• Transpeptidase Active Site Ser attacks Natural Substrate Peptide Bond → Acyl-Enzyme Intermediate = transfers acyl group to neighboring chain → cross-links peptide chain

Bacterial cell wall = unable to synthesize wall → cannot divide → lyses

• Penicillin → kills bacteria = antibiotic

Human cells = NO CELL WALLS = unaffected by penicillin

• Penicillin = one of safest drugs @ recommended doses

What enzymatic function do Transition-State Analogs exploit in irreversible inhibition?

What is an example + enzyme it targets?

Enzymes = catalysts = bind to TS (bonds breaking, bonds forming) tighter than substrate = stabilize TS

• ↓E_A → ↑Rxn rate

• Transition-State Analog = TS-shaped → Enzyme tightly binds to TS analog

Statin = TS Analog → HMG-CoA Reductase

How much stronger do Enzymes bind to TS Analogs than Natural Substrate?

TS Analogs bind covalently to Enzymes: (True/False)

TS Analog drug = x1000 smaller Kd than Natural Substrate

• Enzyme binds to TS Analog x1000 stronger than Natural Substrate

• Tight enough to be functionally irreversible

FALSE → bind through Electrostatic Interactions

What process is HMG-CoA Reductase involved in? Why is it significant?

How are HMG-CoA (substrate) and the Transition State shaped differently?

What shape does statin take?

What enzyme pocket does statin occupy?

What fungus produces statin?

HMG-CoA Reductase = catalyzes 1st step of cholesterol synthesis in liver

• Rate-Limiting Step

HMG-CoA (Substrate) = carbonyl C → trigonal planar

Transition State = negative O → tetrahedral

Statin (drug) = Transition-State Analog = tetrahedral

Statin → TS Pocket NOT active site

Penicillin Citrinum = fungus → statin = defense mechanism against organisms w/ cholesterol-like molecules

Explain the 3 steps of how irreversible inhibitors be used as research tools?

1. Treat Enzyme w/ Radioactively-Labeled Irreversible Inhibitor

2. Find Amino Acid Residue w/ tag

   1. Protease → peptide fragments

   2. Run → radioactively-tagged residue = active site

      1. Does NOT yet prove residue = essential for catalysis

3. Confirm w/ Mutation

   1. Mutate residue → Ala

   2. Retest enzyme

      1. Enzyme activity = decrease → 0

         1. Residue = catalytic → needed to catalyze rxn

      2. Enzyme activity = same

         1. Residue = spectator

What are the 3 components of the Chymotrypsin Active Site?

• Specificity Pocket = chooses substrate

• Catalytic Triad = carries out chemistry

• Oxyanion Hole = stabilizes developing oxygen negative charge

Chymotrypsin, Trypsin, and Elastase prefer the same residues to cleave after: (True/False)

FALSE → Specificity = specific residues chymotrypsin cleaves after

What 2 organs is chymotrypsin produced and activated in?

What type of enzyme is it?

Chymotrypsin = inactive precursor produced in pancreas

• Protease = activated in small intestine → breaks down dietary proteins → smaller protein

What are the 3 cleavage sites of chymotrypsin?

How does substrate recognition explain why these 3 cleavage sites are preferred?

How does this demonstrate Approximation & Orientation?

S1 Pocket supplies the nucleophile: (True/False)

• C-terminals of Phe, Tyr, and Trp

• Substrate Recognition

  • S1 Pocket of Chymotrypsin = deep & hydrophobic

    • Phe, Tyr, and Trp = aromatic ring fit well

  • Allows cleaving @ correct site

FALSE → S1 Pocket CLEAVES bond

What are the 2 cleavage sites of trypsin?

How does substrate recognition explain why these 3 cleavage sites are preferred?

How does this demonstrate Approximation & Orientation?

• Lys, Arg

• S1 Pocket of Trypsin = negatively charged

  • Lys, Arg = positively-charged side chains fit well

  • Allows cleaving @ correct site

What size amino acid residues does elastase prefer to cleave?

• Small residues

• S1 Pocket of Elastase = small

  • Ala, Gly, Val = small side chains fit well

What is the catalytic triad of chymotrypsin?

What 3 amino acids are involved and their general roles?

What 3 enzyme catalytic strategies does Chymotrypsin use and which amino acid residue does it align with?

Catalytic Triad of Chymotrypsin = coordinated set of 3 specific amino acid residues @ Chymotrypsin active site

• Ser195 = Oxygen = Nu

• His57 = proton transfer

• Asp102 = orients His57

Catalytic strategies

• Covalent Catalysis → Ser195

• General Acid-Base Catalysis → His57

• Approximation + Orientation → Asp102

What are the 3 steps of acylation?

1. Nucleophilic Attack

   1. His57 = Base → deprotonates Ser195→ better Nu

   2. Ser195 Oxygen = Nu attacks Carbonyl C of Substrate

2. Tetrahedral Intermediate

   1. C=O bond kicked up to O^- → tetrahedral intermediate

   2. Oxyanion hole = stabilizes O^- through H-Bonds

3. Acyl-Enzyme

   1. His57 = Acid → protonates N-terminal of peptide fragment

   2. N-C bond breaks

      1. N-terminal of peptide fragment = LG

      2. Ester bond = formed b/w Enzyme Ser195 Oxygen + Acyl Fragment

What is the oxyanion hole?

What does it stabilize?

What are the 2 residues that act as H-bond donors?

• The side chains of residues act as H-bond donors: (True/False)

What is the difference between S1 Pocket and Oxyanion Hole?

Oxyanion Hole = preorganized set of H-bond donors of certain enzyme active sites

Stabilizes O^- of tetrahedral intermediate of acylation + deacylation

• Gly193

• Ser195

  • FALSE → Backbone amino groups = H-bond donors

S1 Pocket = recognizes side chain

Oxyanion hole = stabilizes reacting carbonyl oxygen

What are the 3 steps of deacylation?

1. Water Attacks

   1. His57 = Base → deprotonates H₂O→ better Nu

   2. H₂O = Nu attacks Carbonyl C of Ser195 Oxygen + Acyl Fragment

2. Tetrahedral Intermediate

   1. C=O bond kicked up to O^- → tetrahedral intermediate

   2. Oxyanion hole = stabilizes O^- through H-Bonds

3. Enzyme Regeneration

   1. His57 = Acid → protonates Ser195

   2. N-C bond breaks

   3. Carboxyl-Containing Product = LG

   4. Ser195 = restored

What is Burst Kinetics?

What are the 2 phases?

What are 2 things rxn rates are dependent on?

Burst Kinetics = 2-phase rxn pattern

• Burst Phase = Rapid acyl enzyme formation

  • Acylation = fast

  • Enzyme rapidly releases p-nitrophenolate

    • 1 product/enzyme

• Steady-State Phase = Slow enzyme regeneration

  • Deacylation = slower

    • Required for Enzyme Regeneration

  • Rate-determining step

Reaction Rate dependent on:

• Substrate

• Conditions

What are the 3 methods that provide evidence for the Catalytic Triad of Chymotrypsin?

1. Ser195 → DIPF labeling

   1. DIPF = Group-Specific Irreversible Inhibitor = BULKY

      1. Attaches to Ser195 → Enzyme loses function

         1. Chymotrypsin ~28 Serines

   2. Mutation: Ser195 → Ala

      1. Enzyme = unable to form acyl-enzyme

2. His57 → TPCK Labeling

   1. TPCK = Affinity Label Irreversible Inhibitor

      1. Looks like Phe → binds S1 Pocket → BLOCKS His57 from activation of Ser195 + H₂O (proton transfer)

3. Asp102 → Structure + Mutation

   1. Mutation: Asp102 → Asn

      1. ↓Enzyme activity despite:

         1. S1 Pocket

         2. Oxyanion Hole

         3. Nucleophilic Ser195

      2. Asp102 supports His57 → deprotonates + activates Ser195

TPCK must bind to the active site before reaching His57. If Ser195 is already carries DIPF bulky group, will TPCK still label His57?

NO → TPCK will NOT react w/ chymotrypsin (BULKY)