Chapter 6 Enzymes - Lecture notes made by Dr. Shimko of OSU PDF

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Lecture notes made by Dr. Shimko of OSU ...

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2/26/2018

Enzymes Biochem 4511 Figures: Essentials of Biochemistry, 3rd Ed. OSU Custom Edition Principles of Biochemistry 5th Ed., Moran et al. Lehninger Principles of Biochemistry 5th Ed., Nelson & Cox Fundamentals of Biochemistry 2nd Ed., Voet, Voet & Pratt

Enzymes: Nature’s Catalysts •

Catalyst: A substance, usually in small amounts relative to the reactants, that modifies (typically increases) the rate of a reaction without being consumed

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Enzymes are protein catalysts that exhibit: 1) Higher reaction rates - typically 10 6 to 1012 times faster than the uncatalyzed reactions 2) Milder reaction conditions: Below 100 ºC, normal atmospheric pressure, and usually near neutral pH 3) Greater reaction specificity 4) The activity is regulated by concentration of substrates, products, or outside processes (allosteric control, covalent modification, variation of enzyme amount)

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Ribozymes are RNA catalysts

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Enzymes: Vocabulary Words •

Active site: the part of the enzyme which binds the substrate, and contains the residues that directly participate in making and breaking bonds

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Substrate: a reactant, acted on by the enzyme

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Inhibitor: substance that reduces the activity of an enzyme

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Cofactor/coenzyme: small molecule required for some enzyme activity

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Holoenzyme: active enzyme bound to any required cofactors

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Apoenzyme: the enzyme without required cofactor

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Turnover: One step through a reaction cycle

Enzymes Speed Up Reactions

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Specificity: Lock & Key vs. Induced Fit Lock and Key Hypothesis:

Induced Fit Hypothesis:

• Binding site of protein is a perfect match for the substrate

• Binding site of protein is similar to the substrate and when bound, subtle changes occur in the structure of both species

Substrate Specificity •

Specificity and affinity are different properties

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Some enzymes are very specific for one substrate, but most enzymes catalyze reactions for a range of similar substrates: ➢ Alcohol dehydrogenase: Oxidizes small alcohols, but reacts with ethanol faster than methanol or isopropanol

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Some enzymes are very permissive (like digestive enzymes): ➢ Chymotrypsin hydrolyzes peptide bonds at numerous specific recognition sites; can also hydrolyze ester bonds

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Activation Energy and Reaction Coordinates Free Energy of Activation

• In this figure, the transition state is the energetically unfavorable (high energy) complex X‡

Flashback: Thermodynamics vs Kinetics ΔG = ΔH - TΔS

• Thermodynamics is the relationship of reactants to products • If favorable ΔGreaction, a reaction will occur, but only kinetics tells you how quickly!

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Rate Determining Step •

The transition state is not very stable, therefore the amount of transition state in the reaction mixture is very small

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Transition state will quickly decompose back to reactants, or forward to products

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The “rate-determining step” of the overall reaction is the slowest step: typically the formation of the transition state

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Mathematically: reaction rate is proportional to e-ΔG‡/RT

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The larger the value of ΔG‡, the slower the reaction rate

Rate Determining Step: Multistep Reactions •

In a multistep reaction, the step with the largest activation energy (ΔG‡) is the rate limiting step:

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Two different reaction coordinates for conversion of A to P: • •

For blue curve: first step is rate determining For red: second step is rate determining

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Enzymes as Catalysts

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Enzymes reduce ΔG‡ and therefore speed up reactions

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Enzymes provide a reaction pathway with a lower activation energy than the uncatalyzed reaction

Enzyme-Substrate Complex •

How do enzymes lower the activation energy of reactions?

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Mechanisms of action vary, but the first step is the formation of the enzyme-substrate complex (ES complex)

Uncatalyzed:

S

Enzyme catalyzed: E + S

P

ES complex

EP complex

E+P

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Enzyme-Substrate complex

• Multiple weak interactions between enzyme and substrate are responsible for formation of enzyme-substrate complex: Hydrogen bonds Electrostatic interactions Van der Waals forces Hydrophobic interactions

Enzyme-Substrate Complex

• Substrate undergoes reaction while bound to the enzyme

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Rate Enhancement by Enzymes Most enzymes use one or more of the following: 1) Proximity, orientation, and entropy reduction Binding Energy: weak enzyme 2) Preferential binding of the transition state and substrate (transition state stabilization) interactions 3) Acid-base catalysis 4) Covalent catalysis 5) Metal ion catalysis

Interaction with specific catalytic groups

Proximity, Orientation, and Entropy Reduction •

Enzyme-catalyzed reactions use standard organic mechanisms, but enzymes make the reaction more efficient

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Proximity (Local Concentration) and Orientation (proper alignment) are two obvious physical properties that can be manipulated by an enzyme.

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Bringing reactants closer together and aligning them properly for a reaction speeds up encounter rate and overall reaction rate

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Proximity and Entropy Reduction •

Example: bimolecular reaction of imidazole with p-nitrophenylacetate to generate p-nitrophenolate (yellow product)

Intermolecular Reaction

Intramolecular Reaction

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Intramolecular reaction occurs 24 times faster

Orientation 1) Proximity: Enzymes bring substrates into contact with catalytic groups: Typically < 5-fold rate increase 2) Enzymes bind substrates in proper orientations for reaction: Up to 100-fold rate increase Example: SN2 reaction below:

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Proximity, Orientation, and Entropy Reduction

Intramolecular reaction Intramolecular reaction with constrained geometry

Proximity, Orientation, and Entropy Reduction Redux 1) Proximity: Enzymes bring substrates into close vicinity and increase local concentration: Typically < 5-fold rate increase 2) Orientation: Enzymes bind substrates in proper alignment for reactions: Up to 100-fold rate increase. 3) Entropy reduction: Enzymes freeze out substrate rotational and translational motions: Hold the substrate still! a) Rate enhancements: up to 107 in model compounds b) Large entropic penalty that must be made up for in binding energy of the enzyme-substrate complex

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Transition State and Steric Strain •

Enzymes often bind the transition state with greater affinity than either the substrates or the products

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Original concept: Enzymes mechanically strain the substrates towards the transition state geometry

Example: 315-fold rate increase for R=CH3 relative to R=H Strained reactant resembles transition state of the reaction.

Transition State Stabilization Transition State (‡)

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Energy difference (ΔG) for catalyzed reaction (ES‡ - ES) is less than (S‡ - S) for a reaction that is enhanced by transition state stabilization

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Stickase: The Theoretical Enzyme

Preferential Transition State Binding •

Increased preferential binding of the enzyme for the transition state relative to the substrate increases the rate of the catalyzed versus uncatalyzed reaction

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Consequently, enzymes often bind non-reactive molecules which are similar to the transition state much more tightly than they bind active substrates

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The formation of two additional hydrogen bonds between the enzyme and a transition state relative to the substrate results in ~106 rate enhancement

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Transition State Analogs as Inhibitors •

Side effect: A molecule that looks like the transition state of an enzymatic reaction should be a potent inhibitor of the enzyme. Example: Proline Isomerase

Proline Isomerase Catalyzed Reaction

Transition State Analog Inhibitors

Acid-Base Catalysis •

Standard organic arrow reaction mechanisms give hints as to how enzymes might catalyze reactions

Uncatalyzed

Acid catalyzed

Base catalyzed

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Acid-Base Catalysis •

Amino acids often serve as both an acid and base during a single catalytic cycle

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Importantly, this ensures the enzyme is returned to its original state at the end of the reaction allowing the enzyme to process another substrate

Base Catalysis

Acid Catalysis

Regenerated Active Site

Amino Acids in Acid-Base Catalysis

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Amino acids capable of acid-base catalysis are often catalytic residues

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Effects of pH on Enzyme Activity Enzymes are active usually within only a narrow pH range 1) 2) 3) 4)

Binding of substrate to enzyme is dependent on pH Ionization states of catalytic residues are important Ionization of substrate can be important Protein structure may change with extreme pH • Often pH dependence has a bellshaped curve • The inflection points usually indicate the pKa of catalytically important residues • pKas of amino acids in enzyme active sites can vary by several pH units from the expected value

Covalent Catalysis • Enzymes may accelerate reaction rates through transient formation of enzyme-substrate covalent bonds • Typically covalent catalysis involves reaction of a nucleophilic group with an electrophilic substrate • Sometimes referred to as nucleophilic catalysis or electrophilic catalysis depending on the rate-limiting step • Enzyme must be returned to its original state even after forming a covalent bond to allow for multiple enzyme turnovers

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Covalent Catalysis

• Enolate is a fairly high energy transition state, which slows down decarboxylation of acetoacetate to acetone • Conversion of the carbonyl to a Schiff base (imine) would allow the protonated nitrogen to act as better electron sink and consequently lower the transition state energy increasing the reaction rate!

Covalent Catalysis • Example: Decarboxylation of acetoacetate

In this example, RNH2 is a lysine side chain amine

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Covalent Catalysis • Several nucleophiles and electrophiles commonly employed during enzymatic catalysis

Metal Ion Catalysis Functional metal centers play multiple major roles: 1) Binding to substrates to orient them for reactions 2) Oxidation-reduction reactions by changing the metal ion oxidation state 3) Electrostatic stabilization or shielding of negative charges

• Metal ions can make covalently bound water molecules more acidic than free water: good source of catalytic OH–

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Metal Ion Catalysis Example: Carbonic anhydrase, the enzyme that converts CO2 + H2O HCO3- + H+

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Enzyme contains a deep active site cleft with a bound Zn2+

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Catalytic water molecule is activated by the Zn2+ ion

Metal Ion Catalysis 1) Zinc metal center and base catalysis generates nucleophilic OH– 2) Resulting OH– attacks the bound CO2 3) Catalytic site is regenerated by binding and ionization of another H2O

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Serine Proteases • Serine proteases provide a good example of a number of the catalytic mechanisms we have discussed • Serine proteases are very common including chymotrypsin, trypsin, elastase, and many other enzymes • A comparison of various serine proteases provides good examples of substrate specificity and other principles of enzymatic catalysis • As we have discussed, the amide bond is very stable and difficult to break: Uncatalyzed - boil overnight in 6 M HCl Catalyzed - protease activity at physiological pH and temperature

Serine Protease Substrate Preferences • Chymotrypsin, trypsin, and elastase have differing preferences due to amino acids in the substrate binding pockets

Chymotrypsin: aromatic hydrophobic residues Trypsin: Basic residues: Lys, Arg Elastase: Ala, Gly, and Val, but primarily Ala

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Terminology for Protease Recognition • Numbering starts at the cleaved bond: P1 is N-terminal of the cleaved bond and P1’ is C-terminal of the cleaved bond • P denotes the peptide/protein residue and S denotes the enzyme sites that binds to each of these residues • Binding of the P1 residue positions the bond to be cleaved in the active site: proximity and orientation effects

Serine Protease Catalytic Cycle

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Serine Protease Catalytic Cycle

Catalytic Triad

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Catalytic Triad • The catalytic triad is structurally conserved in sequentially unrelated serine proteases

• Convergent evolution: one very good way to activate a hydroxyl group which was arrived at independently by different protein families

Catalytic Triad

• Don’t worry about memorizing the residue numbers, they change from protein to protein, but the roles are maintained • Aspartate hydrogen bonds with histidine which simultaneously hydrogen bonds to serine; precisely aligning the side chains • The strong hydrogen bond between Asp and His means that the proton is shared almost equally • Histidine acts as a general base and removes the proton from the serine hydroxyl group • After deprotonation, the negative charge of Asp stabilizes the positively charged histidine

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Formation of Tetrahedral Intermediate • Substrate binds: proximity, orientation, entropy reduction • Histidine of the catalytic triad acts as a general base, to activate the serine hydroxyl group • Nucleophilic attack of Ser oxygen on the amide carbonyl: covalent catalysis • Transition state stabilization of tetrahedral intermediate in the oxyanion hole

Formation of Tetrahedral Intermediate

• Formation of the tetrahedral intermediate destroys the resonance of the amide bond • Reaction aided by activation of the serine hydroxyl group, orientation of the substrate and nucleophile, and stabilization of tetrahedral intermediate

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Oxyanion hole: Transition State Stabilization

• Oxyanion hole is filled by the tetrahedral intermediate, not by the planar carbonyl of the amide bond • Amide protons from Gly193 and Ser195 make 2 additional hydrogen bonds to the tetrahedral transition state leading to significant stabilization of the ES‡ • Also, additional H-bonding with nitrogen of the amide bond

Formation of Acyl-Enzyme Intermediate

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Deacylation • The cleaved peptide diffuses out of the active site; water diffuses in • Histidine acts as general base to deprotonate water and convert it to hydroxyl ion • Hydroxyl ion attacks acyl-enzyme complex…

Deacylation: Oxyanion Hole Redux • Histidine acts as a general base to deprotonate water converting it to hydroxyl ion • Hydroxyl ion attacks acyl-enzyme complex reforming the tetrahedral oxyanion

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Deacylation: Oxyanion Hole Redux • His acts as general acid to aid in the collapse of the tetrahedral intermediate • Released peptide with newly formed carboxy-terminus diffuses out of the active site to regenerate the enzyme.

Serine Protease Catalytic Cycle

At the end of the cycle, the initial state of the enzyme is regenerated

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