Enzyme Catalysis 28 October 2014 Katja Dove PhD Candidate, Department of Biochemistry, University of Washington 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? Free Energy (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? Free Energy (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? Free Energy (G) Reaction coordinate ΔG < 0, for spontaneous reactions ΔG ǂ  activation energy

What are Enzymes? Proteins that perform biochemical reactions (really fast) Free Energy (G) Reaction coordinate Uncatalyzed (non-enzymatic) reaction: Catalyzed (enzymatic) reaction: R P S + E E  S E  P P + E

What are Enzymes? Proteins that perform biochemical reactions (really fast) Free Energy (G) Reaction coordinate Uncatalyzed (non-enzymatic) reaction: Catalyzed (enzymatic) reaction: R P S + E E  S E  P P + E Terminology S – substrates ( reactants (R) for enzymes) E  S – Enzyme-substrate complex E  P – 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) Free Energy (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 E  S E  P P + E Terminology S – substrates ( reactants (R) for enzymes) E  S – Enzyme-substrate complex E  P – Enzyme-product complex P – products Active site = substrate binding and transformation (aka business end)  specify rearrangement of amino acids

Examples of Speediness Rate 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 Products Regenerated 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

1 2

1 2 Oxyanion hole stabilizes intermediate

1 2 3

4 5

4 5 6

Flash-Back to 1 st step Enzymes are always regenerated! 4 5 6

Metal Ions Cofactors assist in Catalysis: Carbonic anhydrase: H 2 O+CO 2  H + + HCO 3 - 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 Inactivation through covalent bond 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 Inactivation through covalent bond 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 Inactivation through covalent bond 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 Inactivation through covalent bond 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 aspirin naproxen

Aspirin- Mechanism of action HO-CH 2 - Cox-2

Aspirin- Mechanism of action HO-CH 2 - Cox-2 CH 2 - Cox-2 H Acetylated Serine 531

Aspirin- Mechanism of action HO-CH 2 - Cox-2 CH 2 - 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

Enzyme lecture worksheet Which Amino Acids can be used as proton donor/acceptor at physiological pH (~ )? 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 (~ )? 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 diagram A – 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 diagram A – Substrate + Enzyme B - Transition State #1 C – Intermediate #1 D – Transition State #2 E – Intermediate #2 F- Transition State #3 G – Products + Regenerated Enzyme