FUNDAMENTALS OF BIOCHEMISTRY

Fourth EditionDonald Voet - Judith G. Voet - Charlotte W. Pratt

Chapter 11:Enzyme Catalysis

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Enzyme Catalysis

•Enzyme activity and classification

•Enzyme specificity and cofactors

•Transition-State Diagrams

•Catalytic Mechanisms

• Lysozyme

•Serine Proteases

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Enzymes Enzymes differ from ordinary chemical catalyst in several important respects:

• Higher reactions rates: The rates of enzymatically catalyzed reactions are typically several of orders of magnitude greater than those of the corresponding uncatalyzed reactions.

• Milder reactions conditions: Enzymatically catalyzed reactions occur under relative mild conditions (below 100ºC, atmospheric pressure, and nearly neutral pH)

• Greater reaction specificity• Capacity for regulation: The catalytic activities of many enzymes

vary in response to the concentrations of substances other than their substrates.

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Enzymes

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Reaction Coordinate Diagrams

• reactants (substrate, S)• products (P)• enzyme (E)• transition states• ∆G

- spontaneous rxn• activation energy

S

P

E + S ⇌ ES ⇌ E + P

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Reaction Coordinate Diagrams

Catalysts act by providing a reaction pathway with a transition state whose free energy is lower than in the uncatalyzed reaction.

• lowers the activation energy• does not change ∆Grxn

SP

E + S ⇌ ES ⇌ E + P

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Reaction Coordinate DiagramsChemical reactions commonly consist of several steps. For a two-step reaction such as:

A → I → P

there are two transition sates and two activation energy barriers.

The step with the highest transition state acts as a “bottleneck” and is said to be the rate-limiting step.

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Enzyme ClassificationEnzymes are commonly named by appending the suffix -ase to the name of the enzyme's substrate or to a phrase describing the enzyme's catalytic action.

• urease catalyzes the hydrolysis of urea

• alcohol dehydrogenase catalyzes the oxidation of alcohols by removing hydrogen.

There are six major classes of enzymatic reactions.

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Enzyme SpecificitySubstrate-binding site consists of an indentation or cleft on the surface of an enzyme molecule that is complementary in shape to the substrate (geometric complementarity).

Amino acids residues that form the binding site are arranged to specifically attract the substrate (electronic complementarity).

Enzymes act on specific substrates via:• van der Waals• electrostatic• hydrogen bonding• hydrophobic interaction

Nearly all enzymes that participate in chiral reactions are absolutely stereospecific.

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Enzyme SpecificityEnzymes vary in the degree of geometric specificity. A few enzymes are absolutely specific for only one compound.

Most enzymes, however, catalyze the reactions of a small range of related compounds, but with different efficiencies.

• Alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde faster than it oxidizes methanol to formaldehyde (or isopropanol to acetone).

• Digestive enzymes

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Cofactors

Cofactors may be:• metal ions (Cu2+, Fe3+, Zn2+)• organic molecules (coenzymes)

- cosubstrates: NAD+, NADP+

- prosthetic groups are permanently associated with their protein (heme group in myoglobin, hemoglobin and cytochromes)

A catalytically active enzyme-cofactor complex is called a holoenzyme.

An inactive protein resulting from the removal of a holoenzyme’s cofactor is called an apoenzyme.

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How do Enzymes Work?Catalysis can occur through proximity and orientation effects. By simply binding their substrates, enzymes facilitate their catalyzed reactions in 4 ways:

• Enzymes bring substrates into contact with the catalytic groups.• Enzymes bind their substrates in the proper orientation.• Charged groups may help stabilize the transition state of the

reaction (electrostatic catalysis).• Enzymes freeze out the relative translational and rotational motions

of their substrates and catalytic groups.

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How do Enzymes Work?

An enzyme may bind the transition state of the reaction it catalyzes with greater affinity that its substrate or products.

• enzymes that preferentially bind the transition state structure increase its concentration and therefore increase the reaction rate.

- rationale for drug design and enzyme inhibitors

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Catalytic MechanismsThe types of catalytic mechanisms that enzymes employ have been classified as:

• Acid-base catalysis• Metal ion catalysis• Covalent catalysis

• Proximity and orientation effects• Preferential binding of the transition state complex

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Acid-Base CatalysisGeneral acid catalysis is a process in which proton transfer from an acid lowers the free energy of a reaction’s transition state.

• hydrolysis of peptide bonds• phosphate group reactions

The side chains of Asp, Glu, His, Cys, Tyr, and Lys have pK’s in or near physiological pH range, which permits them to act as acid and/or base catalysts.

• activity is sensitive to pH

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Effects of pH on ActivityMost enzymes are active within only a narrow pH range (5-9).

• binding of substrate to enzyme• the ionization states of the amino acid residues• the ionization of the substrate• the variation of protein structure

Inflection point on the curve often provides valuable clues to the identities of the amino acid residues essential for enzymatic activity.

• pK of ~ 4: Asp or Glu• pK of ~6: His• pK of ~10: Lys

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RNase ARNase A is an example of enzymatically mediated acid-base catalysis.

• secreted by the pancreas into the small intestine

• hydrolyzes RNA to its component nucleotides

• 120 amino acids• pI = 9.6

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His 12 (acting as a base) abstracts a proton from an RNA 2’-OH group.This promotes its nucleophilic attack on the adjacent phosphorous atom.His 119 acts as an acid.

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After leaving group departs, water enters the active site, and the 2’,3’-cyclic intermediate is hydrolyzed.

H12 now acts as an acid and H119 as a base.19

Metal Ion CatalysisNearly one-third of all known enzymes require metal ions for catalytic activity.

• metalloenzymes contain tightly bound metal ions (Fe2+, Fe3+, Cu2+, Mn2+, Co2+)

• Na+, K+, Ca2+ play a structural role rather than a catalytic role• Mg2+ and Zn2+ may be either structural or catalytic

Metal ion cofactors:• bind to substrates to orient them properly• mediate oxidation-reduction reactions• electrostatically stabilize or shield negative charges

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Metal Ion CatalysisCarbonic anhydrase:

• catalyzes the rapid conversion of:

CO2 + H2O ⇌ HCO3- + H+

• contains an essential Zn2+ ion (prosthetic group) tetrahedrally coordinated by three His side chains, and 1 H2O molecule

• Zn2+ ion polarizes a water molecule to form OH-, which nucleophilically attacks the substrate CO2 to yield HCO3-.

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Covalent CatalysisCovalent catalysis accelerates reaction rates through the transient formation of a catalyst-substrate covalent bond.

• serine proteases: acyl-serine intermediate• protein kinases and phosphatases: phospho-amino acid interm.

• nucleophilic catalysis: covalent bond is formed by the reaction of a nucleophile group on the catalyst with an electrophilic group on the substrate.

- side chains of His, Cys, Asp, Lys and Ser can participate in covalent catalysis by acting as nucleophiles.

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Covalent CatalysisThe nucleophilicity of a substance is closely related to its basicity.

• biological nucleophiles are negatively charged or contain unshared electron pairs

• biological electrophiles include positively charged groups, contain unfilled valence electron shell, or contain an electronegative atom.

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LysozymeLysozyme is an enzyme that destroys bacteria cell walls.

• hydrolyzing the β(1→4) glycosidic linkage from NAM to NAG in cell wall peptidoglycans.

• occurs widely in the cells of vertebrates- probably functions as bactericidial agent

• example of covalent catalysis and acid-base catalysis.

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Bacterial Cell Wall

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LysozymeLysozyme is an enzyme that destroys bacteria cell walls.

• HEW lysozyme in complex with NAG

• Glu 35 and Asp 52 catalyze the hydrolysis of the glycosidic linkage

- Asp negative charge side chain functions to electronically stabilize the intermediate

- Glu acts as an acid catalyst

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Serine ProteasesSerine proteases are a group of proteolytic enzymes, which include digestive enzymes and specialized proteins that participate in development, blood clotting, inflammation, etc.

• Serine in their active site

Digestive Enzymes: • synthesized by the pancreas and secreted into the small intestine.• catalyze the hydrolysis of peptide bonds (with different specificities)• chymotrypsin: specific for a bulky hydrophobic residue (F, Y, W)• trypsin: specific for a positively charged residue (R, K)• elastase: specific for a small neutral residue (A, G, V)

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Identifying Important Amino AcidsChemical labeling studies helped identify chymotrypsin’s catalytically important groups.

Diisopropylphosphofluoridate (DIPF):• diagnostic test for the presence of the

active site Ser of serine proteases• irreversibly inactivates the enzyme• reacts only with Ser 195 of

chymotrypsin, thereby demonstrating that this residue is the enzyme’s active site Ser.

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Identifying Important Amino AcidsA second catalytically important residue, His 57, was discovered though affinity labeling.

• substrate analog specifically binds at the active site.• tosyl-L-phenylalanine chloromethylketone (TPCK)

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Serine ProteasesChymotrypsin, trypsin and elastase are strikingly similar.

• primary structure: ~40% identical• all have a reactive Ser and a catalytically

essential His

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Serine Proteases

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Mechanism of Serine Proteases

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Serine Proteases - Intermediate

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ZymogensZymogens are inactive precursors of proteolytic enzymes.

• acute pancreatitis• the newly liberated N-term Ile residue

moves from the surface of the protein to an internal position, where its free cationic forms an ion pair with Asp 194.

- without this conformation change, the enzyme cannot properly bind its substrate or stabilize the intermediate

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Objective - Chapter 11• Know what properties distinguish enzymes from other catalysts

• Know how different enzymes are classified

• Be able to explain the factors that influence an enzyme’s substrate specificity

• Know why certain enzymes require cofactors

• Be able to sketch and/or label the various parts of a transition state diagram for a given reaction (with and without a catalyst)

• Know the types of catalytic mechanisms that certain enzymes employ. Be able to describe them in detail (ex: know how protein functional groups can act as acid and base catalysts.)

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Objective - Chapter 11• Know the function of RNase A and how it works

• Know the function of lysozyme and how it works

• Be able to describe how serine proteases work

• Know what distinguishes the three types of serine proteases discussed in class (binding specificity)

• Know the ways that active site residues can be identified

• Know what zymogens are and why there are advantages of synthesizing them.

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