Biochemistry Chapter 6 - Enzyme Catalytic Strategies

Chapter 6 Enzyme Catalytic Strategies

Ch.6 Learning Goals

  • By the end of this chapter, you should be able to:
    1. Discuss four general strategies used by enzymes to accelerate particular reactions.
    2. Give examples of specific chemical features of enzyme active sites that facilitate increasing the rates of specific reactions.
    3. Provide an example of when high absolute rate acceleration is physiologically important and how an enzyme achieves this acceleration.
    4. Understand when high specificity is important for an enzyme and how this specificity is achieved.
    5. Describe an example of when large conformational changes that occur during an enzymatic reaction cycle are used to drive other processes.
    6. Discuss some of the experimental approaches biochemists use to elucidate enzymatic mechanisms.

Section 6.1: Enzymes Use a Core Set of Catalytic Strategies

  • Binding Energy:

    • Definition: Free energy released in the formation of a large number of weak interactions between the enzyme and the substrate.
    • Purposes:
    • Establishes substrate specificity.
    • Increases catalytic efficiency.

  • Induced Fit:

    • Definition: Process by which binding energy promotes structural changes in both the enzyme and the substrate that facilitate catalysis.

Enzymes Use Strategies to Catalyze Specific Reactions (1/2)

  • Covalent Catalysis:

    • Definition: The active site contains a reactive group that becomes temporarily covalently attached to part of the substrate.
    • Example: Proteolytic enzyme chymotrypsin.

  • General Acid–Base Catalysis:

    • Definition: A molecule other than water acts as a proton donor or acceptor.
    • Examples:
    • Histidine residues in chymotrypsin.
    • A phosphate group of ATP substrate for myosin and kinesin.

Enzymes Use Strategies to Catalyze Specific Reactions (2/2)

  • Catalysis by Approximation:

    • Definition: The enzyme brings two substrates together along a single binding surface in a manner that enhances the reaction rate.
    • Example: Carbonic anhydrase.

  • Metal Ion Catalysis:

    • Definition: Metal ions function catalytically, such as by direct coordination, stabilizing negative charges on reaction intermediates, or serving as a bridge between enzyme and substrate.
    • Example: Myosin and kinesin.

Section 6.2: Proteases Facilitate a Fundamentally Difficult Reaction

  • Proteases:
    • Definition: Enzymes that cleave proteins by hydrolysis.
    • Characteristics:
    • Thermodynamically favorable but kinetically extremely slow.

The Chemical Nature of Peptide Bonds Is Responsible for Their Kinetic Stability

  • Peptide Bond Stability:
    • The resonance structure of the peptide bond accounts for its resistance to hydrolysis.
    • The carbonyl carbon involved in the peptide bond is less electrophilic and less susceptible to nucleophilic attack than carbonyl carbons in more reactive compounds.

Chymotrypsin Possesses a Highly Reactive Serine Residue

  • Chymotrypsin:
    • Definition: A proteolytic enzyme that cleaves peptide bonds selectively on the carboxyl-terminal side of large hydrophobic residues.

Chymotrypsin Employs Covalent Catalysis

  • Chymotrypsin uses serine (a powerful nucleophile) to attack the unreactive carbonyl carbon atom of the substrate, becoming covalently attached to the substrate transiently.

Chymotrypsin Action Proceeds in Two Steps Linked by a Covalently Bound Intermediate

  • Chromogenic Substrate:
    • Definition: A substrate analog that forms a colored product measured by light absorbance, used to monitor enzyme kinetics.
    • Example: Chymotrypsin cleaves N-acetyl-L-phenylalanine p-nitrophenyl ester to form yellow p-nitrophenolate.
    • Studies with this substrate revealed catalysis occurs in two stages: rapid (pre-steady state) and slower (steady state).
    • The steps are explained by rapid formation of an acyl-enzyme intermediate and a slower release of the acyl component to regenerate the free enzyme.

Two Kinetic Phases Are Evident by Monitoring the Initial Action of Chymotrypsin

  • Catalysis by chymotrypsin occurs in:
    • Rapid Step (pre-steady state).
    • Slower Step (steady state).

Hydrolysis by Chymotrypsin Involves Covalent Catalysis

  • The two phases are explained by the formation of a covalent enzyme–substrate intermediate.
  • One molecule of p-nitrophenolate is produced rapidly as the acyl-enzyme intermediate is formed.
  • Both phases are required for enzyme turnover.

Serine Is Part of a Catalytic Triad That Also Includes Histidine and Aspartate

  • Chymotrypsin is roughly spherical, containing three polypeptide chains linked by disulfide bonds.
  • Serine 195 marks the active site of chymotrypsin, lying in a cleft on the enzyme's surface.

The Catalytic Triad of Chymotrypsin

  • Catalytic Triad: Set of three amino acid residues in the active site of some enzymes.
    • In chymotrypsin:
    • The side chain of Ser 195 is hydrogen bonded to the imidazole ring of His 57.
    • The –NH group of the His 57 imidazole ring is hydrogen bonded to the carboxylate group of Asp 102.

The Roles of His 57, Ser 195, and Asp 102

  • His 57: Positions the Ser 195 side chain and polarizes serine's hydroxyl group.
    • In the presence of substrate, His 57 acts as a general base catalyst, removing a proton from Ser 195, generating a highly reactive alkoxide ion.
  • Asp 102: Orients His 57 and enhances its ability as a proton acceptor through hydrogen bonding and electrostatic effects.

The Catalytic Triad Increases the Reactivity of Serine 195

  • This structural arrangement effectively enhances the reactivity of Serine 195, allowing it to act as a potent nucleophile.

The Mechanism of Peptide Hydrolysis

  • Oxyanion Hole: Region of the active site that stabilizes unstable tetrahedral reaction intermediates and the transition state preceding the formation of the tetrahedral intermediate.

The Specificity Pocket of Chymotrypsin Favors the Binding of Residues with Long Hydrophobic Side Chains

  • S1 Pocket: Deep hydrophobic pocket into which long, uncharged side chains can fit.
    • Side chain binding positions the adjacent peptide bond for cleavage.
  • Scissile Bond: The bond to be cleaved.

Catalytic Triads Are Found in Other Hydrolytic Enzymes

  • Trypsin and Elastase: Homologs of chymotrypsin with different substrate specificity.
    • Differences arise from small structural differences in the S1 pockets.
  • Other proteases not homologous to chymotrypsin also contain similar active sites that evolved independently.

Specific Cleavage of Polypeptides

  • Table 4.3: Specific cleavage of polypeptides:
    • Cyanogen bromide: Cleaves carboxyl side of methionine residues.
    • O-Iodosobenzoate: Cleaves carboxyl side of tryptophan residues.
    • Hydroxylamine: Cleaves asparagine-glycine bonds.
    • 2-Nitro-5-thiocyanobenzoate: Cleaves amino side of cysteine residues.
    • Enzymatic Cleavage:
    • Trypsin: Carboxyl side of lysine and arginine residues.
    • Clostripain: Carboxyl side of arginine residues.
    • Elastase: Carboxyl side of amino acids with small hydrophobic side chains (valine, alanine, leucine, isoleucine).
    • Thrombin: Carboxyl side of arginine.
    • Chymotrypsin: Carboxyl side of large hydrophobic residues (tyrosine, tryptophan, phenylalanine, leucine, methionine).
    • Carboxypeptidase A: Amino side of C-terminal amino acid (not arginine, lysine, proline).

Some Proteases Cleave Peptides at Other Locations Besides Serine Residues

  • Cysteine Proteases: Rely on a Cys residue, activated by a His residue, to attack the peptide bond as the nucleophile.
  • Aspartyl Proteases: Use a pair of Asp residues that allow a water molecule to attack the peptide bond.
  • Metalloproteases: Use a bound metal ion (typically zinc) activating a water molecule to attack the peptide carbonyl group.

The Catalytic Strategies for Proteases

  • In all three classes, the active site includes features to:
    • Activate a water molecule or another nucleophile.
    • Polarize the peptide carbonyl group.
    • Stabilize a tetrahedral intermediate.

Protease Inhibitors Are Important Drugs

  • Protease Inhibitors: Drugs blocking protease activity.
    • Captopril: Inhibitor of the metalloprotease angiotensin-converting enzyme (ACE), used to regulate blood pressure.
    • Indinavir (Crixivan): Inhibitor of HIV protease (aspartyl protease) that cleaves multidomain viral proteins into active forms, reducing deaths due to AIDS.
    • Designed to resemble peptide substrates of the enzyme.

Section 6.3: Carbonic Anhydrases Make a Fast Reaction Faster

  • Carbon Dioxide: An end product of aerobic metabolism.
  • Carbonic Anhydrases:
    • Convert CO2 into bicarbonate ion and a proton in red blood cells.
    • Dehydrate bicarbonate ion in blood to form CO2 for exhalation.

The Bicarbonate Buffer System

  • pKa Value: Bicarbonate buffer system has a pKa of approximately 6.1 for the reaction of carbonic acid to bicarbonate and H+.
  • Physiological Context: Blood pH is around 7.4. Since pH > pKa, more bicarbonate exists than carbonic acid.
  • Buffering Action:
    • Neutralizing Acids: Carbonic acid can neutralize added acid by dissociating, resisting pH drops.
    • Regulation by Lungs: Carbon dioxide can be expelled by lungs, converting carbonic acid to CO2 and water, effectively removing it.
    • Regulation by Kidneys: Kidneys can adjust bicarbonate excretion or reabsorption.
  • Overall Effectiveness: Dual regulation (lungs for CO2, kidneys for bicarbonate) makes bicarbonate buffer effectively maintain blood pH.

Carbonic Anhydrase Contains a Bound Zinc Ion Essential for Catalytic Activity

  • The human genome contains at least seven homologous carbonic anhydrases.
  • Zn2+ is bound to four ligands: three His residues' imidazole rings and a water molecule (or hydroxide ion, depending on pH).

Catalysis Involves Zinc Activation of a Water Molecule

  • Reaction proceeds at near-maximal rate at pH 8.
  • As pH decreases, reaction rate falls, indicating a group loses a proton at pH 7 (pKa = 7).

Upon Binding to Zinc in Carbonic Anhydrase, the pKa of Water Decreases

  • Zn2+ Effect: Lowers the pKa of water from 15.7 to 7, enabling water to lose a proton at neutral pH, generating the potent nucleophile OH–.

Carbonic Anhydrase Has a CO2 Binding Site Adjacent to the Active Site Zinc Ion

  • A hydrophobic patch serves as the binding site for CO2.

Rapid Regeneration of the Active Form of Carbonic Anhydrase Depends on Proton Availability

  • The zinc-bound water molecule must lose a proton to regenerate the active form of carbonic anhydrase.

The Role of Buffers

  • Highest rates of CO2 hydration require a buffer capable of binding or releasing protons.

The Rate of CO2 Hydration by Carbonic Anhydrase Increases with Buffer Concentration

  • Increasing buffer concentration enhances activity up to a certain point, indicated by kcat vs. buffer concentration graph.

A Proton Shuttle for Large Buffers

  • Proton Shuttle: Transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer.
  • Example: His64 in carbonic anhydrase II.

Section 6.4: Restriction Enzymes Catalyze Highly Specific DNA-Cleavage Reactions

  • Restriction Endonucleases: Enzymes degrades viral DNA by recognizing particular base sequences.
  • Type II Restriction Enzymes: Cleave DNA within recognition sequences.

The Specificity of Restriction Enzymes

  • Must cleave only DNA containing recognition sequences without degrading host DNA.
  • Example: EcoRV recognizes and cleaves the bond between the T and A in the sequence 5′-GATATC-3′.

Cleavage Is by Direct Displacement

  • Restriction enzymes catalyze hydrolysis between the 3′-oxygen atom and phosphorus atom, yielding DNA strands with a free 3′-hydroxyl group and a 5′-phosphoryl group.

Restriction Enzymes Require Magnesium for Catalytic Activity

  • Many enzymes acting on phosphate-containing substrates require Mg2+ or other divalent cations for activity.
  • Role of Magnesium:
    • Coordinated to the protein through two Asp residues, binding water which attacks the phosphorus.

The Complete Catalytic Apparatus Assembled Only Within Cognate DNA Complexes

  • Recognition sequences typically present inverted repeats, giving twofold rotational symmetry.
  • Restriction enzymes are dimers subjected to a 180° rotation.
  • Binding distorts DNA, introducing a kink close to the active site.

The Cognate DNA from the EcoRV Complex Is Substantially Bent

  • Positions the phosphate close to the active site Asp residues, completing a magnesium ion-binding site.

The Binding Energy Drives DNA Distortion

  • Distorted DNA enhances enzyme interactions and increases binding energy, countered by the energy cost of distorting DNA.

Host-Cell DNA Is Protected by Methyl Groups

  • DNA Methylases: Enzymes methylating host DNA on specific adenine bases within recognition sequences to prevent hydrolysis by restriction enzymes.
  • Restriction-Modification System: Consists of a restriction enzyme and corresponding methylase.

Section 6.5: Molecular Motor Proteins Harness Changes in Enzyme Conformation

  • Myosins: Molecular motor proteins catalyzing ATP hydrolysis to generate ADP and inorganic phosphate (Pi), driving molecular motion.

Structure of Myosin

  • Elongated structures with:
    • Globular ATPase domains carrying out ATP hydrolysis.
    • Extended α-helical structures promoting dimer formation.
    • Ancillary associated proteins termed light chains.

Myosin Activity Requires Mg2+ or Mn2+

  • Myosins are inactive without divalent metals (e.g., Mg2+, Mn2+) bonded to ATP.

Formation of the Transition State for ATP Hydrolysis

  • Associated with a significant conformational change in myosin.

Myosin ATPase Transition-State Analog

  • Studied using a stable vanadium-based substrate analog revealing a pentacoordinate transition state.

Water Attack Is Facilitated by Ser 236

  • A water molecule attacks the γ phosphate of ATP, with Ser 236 facilitating proton transfer.

A Substantial Conformation Change Is Associated with Myosin Transition State Formation

  • Changes at the active site amplify large changes in the carboxyl-terminal region of myosin.

The Altered Conformation of Myosin Persists for a Substantial Time

  • Myosin has a turnover number of 1 s−1.
  • Isotopic labeling studies show phosphorus contains oxygen from both ATP and water, indicating reversibility of ATP hydrolysis.

ATP Hydrolysis Is Readily Reversible

  • Studies reveal 2-3 oxygen atoms in phosphate from water, indicating rate-limiting step in hydrolysis is Pi release.

An Actin Filament Has a Polymeric Structure

  • Actin forms filaments for myosin movement, with each monomer containing a bound nucleotide (ATP or ADP).

Scientists Can Watch Single Molecules of Myosin

  • Myosin is fluorescently tagged for localization; inactive without ATP.

Molecular Motor Moves in Discrete Steps

  • Myosin takes steps of approximately 74 nm along actin filaments in the presence of ATP.

Mechanism of Myosin Movement Along Actin Filaments

  • Sequential steps occur during myosin's interaction with ATP and actin, facilitating motion.

A Myosin Assembly from Muscle Has a Two-Headed Structure

  • Comprised of organized assemblies of actin (thin filaments) and multiheaded assemblies of myosin (thick filaments) that lead to muscle contraction.

Actin Networks Can Be Imaged Using Modern Fluorescence Microscopy

  • Cytoskeleton: Internal cellular skeleton made of actin filaments, important for cell shape stability.

Review Questions

  1. What modification is made to DNA that protects the host DNA from cleavage?
    • b. Methylation
  2. Which statement about nonspecific binding to recognition sequences is TRUE?
    • d. For EcoRV, there is a large difference in the binding affinity for cognate and nonspecific DNA.
  3. Which statement about general acid–base catalysis is TRUE?
    • e. In this strategy, anything but water can be used as the donor or acceptor of protons.
  4. Why does chymotrypsin cleave peptide bonds only after amino acids with aromatic or large hydrophobic side chains?
    • a. Its active site is preceded by a specificity hydrophobic pocket, which binds such side chains.