Enzyme Catalysis28 October 2014

Katja DovePhD Candidate,

Department of Biochemistry, University of Washington

Email: Katja.Dove@seattlecolleges.edu

• Please turn in your take-home part for midterm 1 BEFORE class

• I will hand back in-class exams at the end of class today

• NO class on THURSDAY 10/30/14, Jim will be back NEXT week and he will continue with Chapter 12 (Enzyme Kinetics)

How long would it take you to digest a fried breakfast without the help of digestive enzymes?

A. 1 day

B. 1 week

C. 1 month

D. 1 year

E. 50 years

F. Never

How long would it take you to digest a fried breakfast without the help of digestive enzymes?

A. 1 day

B. 1 week

C. 1 month

D. 1 year

E. 50 years

F. Never

How long would it take you to digest a fried breakfast without the help of digestive enzymes?

A. 1 day

B. 1 week

C. 1 month

D. 1 year

E. 50 years

F. Never

What is a “spontaneous” reaction?

How long would it take you to digest a fried breakfast without the help of digestive enzymes?

A. 1 day

B. 1 week

C. 1 month

D. 1 year

E. 50 years

F. Never

What is a “spontaneous” reaction?

Fre

e E

nerg

y (G

)

Reaction coordinate

How long would it take you to digest a fried breakfast without the help of digestive enzymes?

A. 1 day

B. 1 week

C. 1 month

D. 1 year

E. 50 years

F. Never

What is a “spontaneous” reaction?

Fre

e E

nerg

y (G

)

Reaction coordinate

ΔG < 0, for spontaneous reactions

How long would it take you to digest a fried breakfast without the help of digestive enzymes?

A. 1 day

B. 1 week

C. 1 month

D. 1 year

E. 50 years

F. Never

What is a “spontaneous” reaction?

Fre

e E

nerg

y (G

)

Reaction coordinate

ΔG < 0, for spontaneous reactions

ΔGǂ activation energy

What are Enzymes? Proteins that perform biochemical reactions (really fast)

Fre

e E

nerg

y (G

)

Reaction coordinate

Uncatalyzed (non-enzymatic) reaction:

Catalyzed (enzymatic) reaction:

R P

S + E ES EP P + E

What are Enzymes? Proteins that perform biochemical reactions (really fast)

Fre

e E

nerg

y (G

)

Reaction coordinate

Uncatalyzed (non-enzymatic) reaction:

Catalyzed (enzymatic) reaction:

R P

S + E ES EP P + E

Terminology• S – substrates ( reactants (R) for enzymes)• ES – Enzyme-substrate complex• EP – Enzyme-product complex• P – products• Active site = substrate binding and transformation (aka

business end) specify rearrangement of amino acids

What are Enzymes? Proteins that perform biochemical reactions (really fast)

Fre

e E

nerg

y (G

)

Reaction coordinate

Enzymes = biological catalysts speed up reactions by lowering

activation Energy

DO NOT make reactions spontaneous (e.i. no influence on thermodynamics, but “only” kinetics)

Uncatalyzed (non-enzymatic) reaction:

Catalyzed (enzymatic) reaction:

R P

S + E ES EP P + E

Terminology• S – substrates ( reactants (R) for enzymes)• ES – Enzyme-substrate complex• EP – Enzyme-product complex• P – products• Active site = substrate binding and transformation (aka

business end) specify rearrangement of amino acids

Examples of SpeedinessRate enhancement = rate catalyzed/rate uncatalyzed

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions • Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions • Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

Two-Step Reaction:

Which one is the rate-limiting step?

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions • Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions– Temp below 100°C, neutral pH, atmospheric pressure

• Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions– Temp below 100°C, neutral pH, atmospheric pressure

• Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions– Temp below 100°C, neutral pH, atmospheric pressure

• Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions– Temp below 100°C, neutral pH, atmospheric pressure

• Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions– Temp below 100°C, neutral pH, atmospheric pressure

• Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions– Temp below 100°C, neutral pH, atmospheric pressure

• Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

General Properties of Enzymes• Increase reaction rates

– lowering the activation energy

• High specificity – substrate binding (“Key & Lock”)– Stereospecific

• Mild reaction conditions– Temp below 100°C, neutral pH, atmospheric pressure

• Regenerated (not used up)• Cofactors (e.g. metals)• Capacity for regulation (e.g. glycolysis)• Drug targets (e.g. NSAIDs, antibiotics)

Catalytic mechanisms

• Covalent • Acid/Base• Metal ions• Proximity and orientation of reactants

Naming convention: -ases

Covalent Catalysis

• Covalent mechanisms often need a nucleophile• uncatalyzed: A B A + B• catalyzed: A B + :X A X + B A + B + :X

(:X)

Covalent Catalysis

• Covalent mechanisms often need a nucleophile• uncatalyzed: A B A + B• catalyzed: A B + :X A X + B A + B + :X

(:X)

Covalent Catalysis

• Covalent mechanisms often need a nucleophile• uncatalyzed: A B A + B• catalyzed: A B + :X A X + B A + B + :X

(:X)

Covalent Catalysis

• Covalent mechanisms often need a nucleophile• uncatalyzed: A B A + B• catalyzed: A B + :X A X + B A + B + :X

(:X)

Acid-Base catalysis• Enzymes use amino acid residues as proton acceptors/donor

to aid in catalysis

• Covalent catalysis: A B + :X A X + B A + B + :X• Acid-Base catalysis:

A B + X H + :Y A X + B +Y H A + B + X H :Y+H+

substrate

Nucleophile/acid

base

Enzyme-intermediate complex

Product #1

Conjugated base

Products

Regenerated active site of enzyme

Acid-Base catalysis• Enzymes use amino acid residues as proton acceptors/donor

to aid in catalysis

• Covalent catalysis: A B + :X A X + B A + B + :X• Acid-Base catalysis:

A B + X H + :Y A X + B +Y H A + B + X H :Y+H+

substrate

Nucleophile/acid

base

Enzyme-intermediate complex

Product #1

Conjugated base

Products

Regenerated active site of enzyme

Acid-Base catalysis• Enzymes use amino acid residues as proton acceptors/donor

to aid in catalysis

• Covalent catalysis: A B + :X A X + B A + B + :X• Acid-Base catalysis:

A B + X H + :Y A X + B +Y H A + B + X H :Y+H+

substrate

Nucleophile/acid

base

Enzyme-intermediate complex

Product #1

Conjugated base

Products

Regenerated active site of enzyme

Acid-Base catalysis• Enzymes use amino acid residues as proton acceptors/donor

to aid in catalysis

• Covalent catalysis: A B + :X A X + B A + B + :X• Acid-Base catalysis:

A B + X H + :Y A X + B +Y H A + B + X H :Y+H+

substrate

Nucleophile/acid

base

Enzyme-intermediate complex

Product #1

Conjugated base

Products

Regenerated active site of enzyme

Acid-Base catalysis• Enzymes use amino acid residues as proton acceptors/donor

to aid in catalysis

• Covalent catalysis: A B + :X A X + B A + B + :X• Acid-Base catalysis:

A B + X H + :Y A X + B +Y H A + B + X H :Y+H+

substrate

Nucleophile/acid

base

Enzyme-intermediate complex

Product #1

Conjugated acid

Products

Regenerated active site of enzyme

Acid-Base catalysis• Enzymes use amino acid residues as proton acceptors/donor

to aid in catalysis

• Covalent catalysis: A B + :X A X + B A + B + :X• Acid-Base catalysis:

A B + X H + :Y A X + B +Y H A + B + X H :Y+H+

substrate

Nucleophile/acid

base

Enzyme-intermediate complex

Product #1

Conjugated acid

ProductsRegenerated active site of enzyme

Acid/Base reaction with Covalent Intermediate: Serine proteases

Proteases

• Also called peptidase or proteinase

• Perform proteolysis = breakdown of peptides by hydrolysis of peptides bonds

• Serine proteases – Serine acts as nucleophile

• Catalytic triad• Stabilize transition state

(oxyanion hole)

1 2

Catalytic triad

1 2

Catalytic triad

1 2

Catalytic triad

1 2

Oxyanion hole stabilizes intermediate

1 2

3

1 2

34

4

5

4

56

Flash-Back to 1st step

Enzymes are always regenerated!

4

56

Metal Ions Cofactors assist in Catalysis:Carbonic anhydrase: H2O+CO2 H+ + HCO3

-

Metal Ions can:

• Bind and orient substrates• Stabilize charged intermediate• Perform oxidation/reduction chemistry

Enzyme Inhibition

• Many therapeutic drugs are enzyme inhibitors• Enzyme kinetics important to drug design

(effectiveness)• Natural toxins are also enzyme inhibitors• Often Enzyme inhibitors either

prevent/interfere with substrate binding OR lower catalytic activity of enzyme OR both

Classes of enzyme inhibitors

Reversible • Competitive

– Inhibitor looks like substrate• HIV protease inhibitors

• Mixed• Non-competitive

Irreversible • “inactivators”• “suicide” inhibitors

– Covalent modification enzyme-inhibitor complex • Aspirin

Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors)

“bad” - toxic “good” - antibiotics

Diisopropylfluorophosphate (DIFP) penicillin

Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine)

Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

Enzyme

Enzyme

Inactivation through covalent bond

Enzyme

Enzyme

Inactivation through covalent bond

Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors)

“bad” - toxic “good” - antibiotics

Diisopropylfluorophosphate (DIFP) penicillin

Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine)

Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

Enzyme

Enzyme

Inactivation through covalent bond

Enzyme

Enzyme

Inactivation through covalent bond

Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors)

“bad” - toxic “good” - antibiotics

Diisopropylfluorophosphate (DIFP) penicillin

Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine)

Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

Enzyme

Enzyme

Inactivation through covalent bond

Enzyme

Enzyme

Inactivation through covalent bond

Irreversible Enzyme Inhibition – mechanism based (suicide inhibitors)

“bad” - toxic “good” - antibiotics

Diisopropylfluorophosphate (DIFP) penicillin

Enzyme = acetylcholinesterase (hydrolase that hydrolysis acetylcholine)

Enzyme = transpeptidase (catalysis cross-linkages in peptidoglycan cell walls)

Enzyme

Enzyme

Inactivation through covalent bond

Enzyme

Enzyme

Inactivation through covalent bond

49

adds 2 oxygen molecules to arachidonic acid to make prostaglandin

pulls one molecule of arachidonic acid out of membrane

pain and inflammation

How can NSAIDs prevent pain and inflammation?

How NSAIDs work by inhibiting COX-2

aspirinnaproxen

Aspirin- Mechanism of action

HO-CH2- Cox-2

Aspirin- Mechanism of action

HO-CH2- Cox-2

CH2- Cox-2 H Acetylated Serine 531

Aspirin- Mechanism of action

HO-CH2- Cox-2

CH2- Cox-2 H

Blocks substrate binding no production of prostaglandins no pain/inflammation

Acetylated Serine 531

Side effects of NSAIDs: COX-1

• COX-2 has a “sibling” called COX-1• Look similar, some COX-2 drugs also bind COX-

1 which leads to off-target effects

http://tr-i-life.tumblr.com/post/32829231019/quick-pharm-review-aspirin-vs-ibuprofen

Enzyme lecture worksheet• Which Amino Acids can be used as proton donor/acceptor at physiological

pH (~7.0-7.4)? Draw each side chain and indicate whether or not it could be either act as a proton donor or acceptor.

• Which Amino Acids side chains could act as an electrophile at physiological pH (~7.0-7.4)? Which ones are more LIKELY to be a nucleophile? Draw each side chain and mark the nucleophile with an arrow.

• Label the reaction coordinate diagram. Substrate(s), Product(s) Where are the intermediates? Where are the transition states? Which one is the rate limiting step? Is this reaction spontaneous? Why?

Proton acceptors/donors

Nucleophiles

Label the reaction coordinate diagramA – Substrate + Enzyme

B - Transition State #1

C – Intermediate #1

D – Transition State #2

E – Intermediate #2

F- Transition State #3

G – Products + Regenerated Enzyme

D is the rate limiting Step

Spontaneous reactions because dG is negative

Label the reaction coordinate diagramA – Substrate + Enzyme

B - Transition State #1

C – Intermediate #1

D – Transition State #2

E – Intermediate #2

F- Transition State #3

G – Products + Regenerated Enzyme