Chapter 14 Mechanisms of Enzyme Action

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Chapter 14Mechanisms of Enzyme Action
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Outline

• What are the magnitudes of enzyme-induced rate
  accelerations?
• What role does transition-state stabilization
  play in enzyme catalysis?
• How does destabilization of ES affect enzyme
  catalysis?
• How tightly do transition-state analogs bind to
  the active site?
• What are the mechanisms of catalysis?
• What can be learned from typical enzyme
  mechanisms?

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14.1 What Are the Magnitudes of Enzyme-Induced
Rate Accelerations?

• Enzymes are powerful catalysts
• The large rate accelerations of enzymes (107 to
  1015) correspond to large changes in the free
  energy of activation for the reaction
• All reactions pass through a transition state on
  the reaction pathway
• The active sites of enzymes bind the transition
  state of the reaction more tightly than the
  substrate
• By doing so, enzymes stabilize the transition
  state and lower the activation energy of the
  reaction

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14.2 What Role Does Transition-State
Stabilization Play in Enzyme Catalysis?

• The catalytic role of an enzyme is to reduce the
  energy barrier between substrate S and transition
  state X
• Rate acceleration by an enzyme means that the
  energy barrier between ES and EX must be smaller
  than the barrier between S and X
• This means that the enzyme must stabilize the EX
  transition state more than it stabilizes ES

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14.2 What Role Does Transition-State
Stabilization Play in Enzyme Catalysis?
Enzymes catalyze reactions by lowering the
activation energy. Here the free energy of
activation for (a) the uncatalyzed reaction is
larger than that of the enzyme-catalyzed reaction.
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14.2 What Role Does Transition-State
Stabilization Play in Enzyme Catalysis?

• Competing effects determine the position of ES on
  the energy scale
• Try to mentally decompose the binding effects at
  the active site into favorable and unfavorable
• The binding of S to E must be favorable
• But not too favorable!
• Km cannot be "too tight" - goal is to make the
  energy barrier between ES and EX small

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14.3 How Does Destabilization of ES Affect Enzyme
Catalysis?

• Raising the energy of ES raises the rate
• For a given energy of EX, raising the energy of
  ES will increase the catalyzed rate
• This is accomplished by
• a) loss of entropy due to formation of ES
• b) destabilization of ES by
• strain
• distortion
• desolvation

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14.3 How Does Destabilization of ES Affect Enzyme
Catalysis?
(a) Catalysis does not occur if ES and X are
equally stabilized. (b) Catalysis will occur if
X is stabilized more than ES.
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14.3 How Does Destabilization of ES Affect
Enzyme Catalysis?
(a) Formation of the ES complex results in
entropy loss. The ES complex is a more highly
ordered, low-entropy state for the substrate.
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14.3 How Does Destabilization of ES Affect Enzyme
Catalysis?
(b) Substrates typically lose waters of hydration
in the formation in the formation of the ES
complex. Desolvation raises the energy of the ES
complex, making it more reactive.
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14.3 How Does Destabilization of ES Affect
Enzyme Catalysis?
(c) Electrostatic destabilization of a substrate
may arise from juxtaposition of like charges in
the active site. If charge repulsion is relieved
in the reaction, electrostatic destabilization
can result in a rate increase.
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14.4 How Tightly Do Transition-State Analogs Bind
to the Active Site?

• Very tight binding to the active site
• The affinity of the enzyme for the transition
  state may be 10 -20 to 10-26 M!
• Can we see anything like that with stable
  molecules?
• Transition state analogs (TSAs) are stable
  molecules that are chemically and structurally
  similar to the transition state
• Proline racemase was the first case

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14.4 How Tightly Do Transition-State Analogs Bind
to the Active Site?
The proline racemase reaction. Pyrrole-2-carboxyla
te and ?-1-pyrroline-2-carboxylate mimic the
planar transition state of the reaction.
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Transition-State Analogs Make Our World Better

• Enzymes are often targets for drugs and other
  beneficial agents
• Transition-state analogs often make ideal enzyme
  inhibitors
• Enalapril lowers blood pressure
• Statins lower serum cholesterol
• Protease inhibitors are AIDS drugs
• Juvenile hormone esterase is a pesticide target
• Tamiflu is a viral neuraminidase inhibitor

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How many other drug targets might there be?

• The human genome contains approximately 20,000
  genes
• How many might be targets for drug therapy?
• More than 3000 experimental drugs are presently
  under study and testing
• These and many future drugs will be designed as
  transition-state analog inhibitors
• See the DrugBank http//drugbank.ca

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14.5 What Are the Mechanisms of Catalysis?

• Enzymes facilitate formation of near-attack
  complexes
• Protein motions are essential to enzyme catalysis
• Covalent catalysis
• General acid-base catalysis
• Low-barrier hydrogen bonds
• Metal ion catalysis

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Enzymes facilitate formation of near-attack
complexes

• X-ray crystal structure studies and computer
  modeling have shown that the reacting atoms and
  catalytic groups are precisely positioned for
  their roles
• Such preorganization selects substrate
  conformations in which the reacting atoms are in
  van der Waals contact and at an angle resembling
  the bond to be formed in the transition state
• Thomas Bruice has termed such arrangements
  near-attack conformations (NACs)
• NACs are precursors to reaction transition states

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Enzymes facilitate formation of near-attack
complexes

• Thomas Bruice has proposed that near-attack
  conformations are precursors to transition states
• In the absence of an enzyme, potential reactant
  molecules adopt a NAC only about 0.0001 of the
  time
• On the other hand, NACs have been shown to form
  in enzyme active sites from 1 to 70 of the time

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Enzymes facilitate formation of near-attack
complexes
Figure 14.7 NACs are characterized as having
reacting atoms within 3.2 Å and an approach angle
of 15 of the bonding angle in the transition
state.
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Figure 14.8 The active site of liver alcohol
dehydrogenase a near-attack complex.
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Protein Motions Are Essential to Enzyme Catalysis

• Proteins are constantly moving bonds vibrate,
  side chains bend and rotate, backbone loops
  wiggle and sway, and whole domains move as a unit
• Enzymes depend on such motions to provoke and
  direct catalytic events
• Protein motions support catalysis in several
  ways. Active site conformation changes can
• Assist substrate binding
• Bring catalytic groups into position
• Induce formation of NACs
• Assist in bond making and bond breaking
• Facilitate conversion of substrate to product

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Covalent Catalysis

• Some enzymes derive much of their rate
  acceleration from formation of covalent bonds
  between enzyme and substrate
• The side chains of amino acids in proteins offer
  a variety of nucleophilic centers for catalysis
• These groups readily attack electrophilic centers
  of substrates, forming covalent enzyme-substrate
  complexes
• The covalent intermediate can be attacked in a
  second step by water or by a second substrate,
  forming the desired product

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Covalent Catalysis
Examples of covalent enzyme-substrate
intermediates.
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Covalent Catalysis
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General Acid-Base Catalysis

• Catalysis in which a proton is transferred in the
  transition state
• "Specific" acid-base catalysis involves H or OH-
  that diffuses into the catalytic center
• "General" acid-base catalysis involves acids and
  bases other than H and OH-
• These other acids and bases facilitate transfer
  of H in the transition state

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General Acid-Base Catalysis
Catalysis of p-nitrophenylacetate hydrolysis can
occur either by specific acid hydrolysis or by
general base catalysis.
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Low-Barrier Hydrogen Bonds (LBHBs)

• The typical H-bond strength is 10-30 kJ/mol, and
  the O-O separation is typically 0.28 nm
• As distance between heteroatoms becomes smaller
  (lt0.25 nm), H bonds become stronger
• Stabilization energies can approach 60 kJ/mol in
  solution
• pKa values of the two electronegative atoms must
  be similar
• Energy released in forming an LBHB can assist
  catalysis

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Low-Barrier Hydrogen Bonds (LBHBs)
Energy diagrams for conventional H bonds (a), and
low-barrier hydrogen bonds (b and c). In (c),
the O-O distance is 0.23 to 0.24 nm, and bond
order for each O-H interaction is 0.5.
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Quantum Mechanical Tunneling

• Tunneling provides a path around the usual
  energy of activation for steps in chemical
  reactions
• Many enzymes exploit this
• According to quantum theory, there is a finite
  probability that any particle can appear on the
  other side of an activation barrier for a
  reaction step
• The likelihood of tunneling depends on the
  distance over which a particle must move
• Only protons and electrons have a significant
  probability of tunneling

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Quantum Mechanical Tunneling

• The de Broglie equation relates the de Broglie
  wavelength to the mass and energy of a particle
• Tunneling can only play a significant role in a
  reaction when the wavelength of the transferring
  particle is similar to the distance over which it
  is transferred
• de Broglie wavelengths for protons and electrons
  are 0.9Å and 38Å, respectively
• Tunneling probably contributes to most, if not
  all, hydrogen transfer reactions

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Tunneling between donor and acceptor
If the distance for particle transfer is
sufficiently small, overlap of probability
functions (red) permit efficient quantum
mechanical tunneling between donor (D) and
acceptor (A)
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Metal Ion Catalysis
Thermolysin is an endoprotease with a catalytic
Zn2 ion in the active site. The Zn2 ion
stabilizes the buildup of negative charge on the
peptide carbonyl oxygen, as a glutamate residue
deprotonates water, promoting hydroxide attack on
the carbonyl carbon.
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How Do Active-Site Residues Interact to Support
Catalysis?

• About half of the amino acids engage directly in
  catalytic effects in enzyme active sites
• Other residues may function in secondary roles in
  the active site
• Raising or lowering catalytic residue pKa values
• Orientation of catalytic residues
• Charge stabilization
• Proton transfers via hydrogen tunneling

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14.5 What Can Be Learned From Typical Enzyme
Mechanisms?

• First Example the serine proteases
• Enzyme and substrate become linked in a covalent
  bond at one or more points in the reaction
  pathway
• The formation of the covalent bond provides
  chemistry that speeds the reaction
• Serine proteases also employ general acid-base
  catalysis

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The Serine Proteases

• Trypsin, chymotrypsin, elastase, thrombin,
  subtilisin, plasmin, TPA
• All involve a serine in catalysis - thus the name
  
• Ser is part of a "catalytic triad" of Ser, His,
  Asp
• Serine proteases are homologous, but locations of
  the three crucial residues differ somewhat
• Enzymologists agree, however, to number them
  always as His57, Asp102, Ser195
• Burst kinetics yield a hint of how they work

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The Catalytic Triad of the Serine Proteases
Structure of chymotrypsin (white) in a complex
with eglin C (blue ribbon structure), a target
substrate. His57 (red) is flanked by Asp102
(gold) and Ser195 (green). The catalytic site is
filled by a peptide segment of eglin. Note how
close Ser195 is to the peptide that would be
cleaved in the reaction.
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The Catalytic Triad of the Serine Proteases
The catalytic triad at the active site of
chymotrypsin (and the other serine proteases.)
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Serine Protease Binding Pockets are Adapted to
Particular Substrates
The substrate-binding pockets of trypsin,
chymotrypsin, and elastase. Asp189 (aqua)
coordinates Arg and Lys residues of substrates in
the trypsin pocket. Val216 (purple) and Thr226
(green) make the elastase pocket shallow and able
to accommodate only small, nonbulky residues. The
chymotrypsin pocket is hydrophobic.
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Serine Proteases Cleave Simple Organic Esters,
such as p-Nitrophenylacetate
Chymotrypsin cleaves simple esters, in addition
to peptide bonds. p-Nitrophenylacetate has been
used in studies of the chymotrypsin mechanism.
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Serine Protease Mechanism

• A mixture of covalent and general acid-base
  catalysis
• Asp102 functions only to orient His57
• His57 acts as a general acid and base
• Ser195 forms a covalent bond with peptide to be
  cleaved
• Covalent bond formation turns a trigonal C into a
  tetrahedral C
• The tetrahedral oxyanion intermediate is
  stabilized by the backbone N-H groups of Gly193
  and Ser195

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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism binding
of a model substrate.
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism the
formation of the covalent ES complex (E-Ser195S
complex) involves general base catalysis by His57
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism His57
stabilized by a LBHB.
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism collapse
of the tetrahedral intermediate releases the
first product.
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism The
amino product departs, making room for an
entering water molecule.
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism
Nucleophilic attack by water is facilitated by
His57, acting as a general base.
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism Collapse
of the tetrahedral intermediate cleaves the
covalent intermediate, releasing the second
product.
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism Carboxyl
product release completes the serine protease
mechanism.
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The Serine Protease Mechanism in Detail
Figure 14.21 The chymotrypsin mechanism At the
completion of the reaction, the side chains of
the catalytic triad are restored to their
original states.
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Transition-State Stabilization in the Serine
Proteases

• The chymotrypsin mechanism involves two
  tetrahedral oxyanion transition states
• These transition states are stabilized by a pair
  of amide groups that is termed the oxyanion
  hole
• The amide N-H groups of Ser195 and Gly193 provide
  primary stabilization of the tetrahedral oxyanion

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The oxyanion hole
The oxyanion hole of chymotrypsin stabilizes the
tetrahedral oxyanion transition state seen in the
mechanism of Figure 14.21.