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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14.1 What Are the Magnitudes of Enzyme-Induced
Rate Accelerations?
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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.
• 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.
• See Eq. 14.3.

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14.2 What Role Does Transition-State
Stabilization Play in Enzyme Catalysis?
Figure 14.1 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 should not 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 or distortion.
• Desolvation.
• Electrostatic effects.

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14.3 How Does Destabilization of ES Affect Enzyme
Catalysis?
Figure 14.2 The intrinsic binding energy of ES
is compensated by entropy loss due to binding of
E and S and by destabilization due to strain and
distortion.
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14.3 How Does Destabilization of ES Affect Enzyme
Catalysis?
Figure 14.3(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?
Figure 14.4(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?
Figure 14.4(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?
Figure 14.4(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.
• See Figure 14.6 for some recent cases.

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14.4 How Tightly Do Transition-State Analogs Bind
to the Active Site?
Figure 14.5 The proline racemase reaction.
Pyrrole-2-carboxylate and ?-1-pyrroline-2-carboxy
late mimic the planar transition state of the
reaction.
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14.4 How Tightly Do Transition-State Analogs Bind
to the Active Site?
Figure 14.6(a) Phosphoglycolohydroxamate is an
analog of the enediolate transition state of the
yeast aldolase reaction.
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14.4 How Tightly Do Transition-State Analogs Bind
to the Active Site?
(b) Purine riboside inhibits adenosine deaminase.
The hydrated form is an analog of the 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 and Aliskiren lower 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 to read and write mechanisms

• The custom of writing chemical reaction
  mechanisms with electron dots and curved arrows
  began with Gilbert Newton Lewis and Sir Robert
  Robinson.
• Review of Lewis dot structures, the concepts of
  valence electrons and formal charge.
• Formal charge group number nonbonding
  electrons (1/2 shared electrons).
• Electronegativity is also important
• F gt O gt N gt C gt H.
• The arrows used always point from where electrons
  originate to where they are going.

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How to read and write mechanisms
For a bond-breaking event, the arrow begins in
the middle of the bond, and the arrow points to
the atom that will accept the electrons.
For a bond-making event, the arrow begins at the
source of the electrons (for example, a nonbonded
pair), and the arrowhead points to the atom where
the new bond will be formed.
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How to read and write mechanisms

• It has been estimated that 75 of the steps in
  enzyme reaction mechanisms are proton (H)
  transfers.
• If the proton is donated or accepted by a group
  on the enzyme, it is often convenient (and
  traditional) to represent the group as B, for
  base, even if B is protonated and behaving as
  an acid

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How to read and write mechanisms

• It is important to appreciate that a proton
  transfer can change a nucleophile into an
  electrophile, and vice versa.
• Thus, it is necessary to consider
• The protonation states of substrate and
  active-site residues (mostly the sidechains).
• How pKa values of sidechains can change in the
  environment of the active site.
• For example, an active-site histidine, which
  might normally be protonated, can be deprotonated
  by another group and then act as a base,
  accepting a proton from the substrate.

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How to read and write mechanisms
An active-site histidine, which might normally be
protonated, can be deprotonated by another group
and then act as a base, accepting a proton from
the substrate.
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How to read and write mechanisms
Water can often act as an acid or base at the
active site through proton transfer with an
assisting active-site residue
This type of chemistry is the basis for general
acid-base catalysis (discussed on pages 430-431).
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14.5 What Are the Mechanisms of Catalysis?

• Enzymes facilitate formation of near-attack
  complexes (NAC).
• 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).

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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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Enzymes facilitate formation of near-attack
complexes
Figure 14.7 In an enzyme active site, the NAC
forms more readily than in the uncatalyzed
reaction. The energy separation between the NAC
and the transition state is approximately the
same in the presence and absence of the enzyme.
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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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Protein Motions Are Essential to Enzyme Catalysis
Figure 14.9 Human cyclophilin A is a prolyl
isomerase, which catalyzes the interconversion
between trans and cis conformations of proline in
peptides.
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Protein Motions Are Essential to Enzyme Catalysis
Figure 14.9 The active site of cyclophilin with
a bound peptide containing proline in cis and
trans conformations. Motion by active site
residues promote catalysis in cyclophilin.
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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
Figure 14.11 Examples of covalent bond formation
between enzyme and substrate. A nucleophilic
center X on an enzyme attacks a phosphorus atom
to form a phosphoryl enzyme intermediate.
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Covalent Catalysis
Figure 14.11 Examples of covalent bond formation
between enzyme and substrate. A nucleophilic
center X on an enzyme attacks a carbonyl C to
form an acyl enyzme intermediate.
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Covalent Catalysis
Figure 14.11 Examples of covalent bond formation
between enzyme and substrate. A nucleophilic
center X attacks the anomeric carbon of a
glycoside, forming a glucosyl enzyme intermediate.
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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.
• See Figure 14.12.

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General Acid-base Catalysis
Figure 14.12 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)
Figure 14.13 Energy diagrams for conventional H
bonds (a), and low-barrier hydrogen bonds (b and
c).s 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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Metal Ion Catalysis
Figure 14.14 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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How Do Active-Site Residues Interact to Support
Catalysis?
The active site of aromatic amine dehydrogenase,
showing the relationship of Asp128, Thr172, and
Cys171. Coupling of local motions of these
residues to vibrational states involved in proton
transfer contributes to catalysis.
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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 Serine Proteases
Figure 14.15 The amino acid sequences of
chymotrypsinogen, trypsin, and elastase.
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The Catalytic Triad of the Serine Proteases
Figure 14.16 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
Figure 14.17 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
Figure 14.18 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
Figure 14.19 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 Proteases Display Burst Kinetics
Figure 14.20 Burst kinetics in the chymotrypsin
reaction. p-NO2-phenol Absorbs at 400
nm. Covalent catalysis ?
Acyl enzyme
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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 peptide N-H of Gly193 and of
  Ser195 in what is referred to as an oxyanion
  hole.

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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 involves
general base catalysis by His57.
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The Serine Protease Mechanism in Detail
oxyanion
Figure 14.21 The chymotrypsin mechanism His57
stabilized by a LBHB. The tetrahedral
intermediate in this diagram is an oxyanion.
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The Serine Protease Mechanism in Detail
oxyanion
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, leaving the acylated
enzyme and 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
oxyanion
Figure 14.21 The chymotrypsin mechanism A
second oxyanion from attack by water. 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 intermediates.
• These intermediates are stabilized by a pair of
  amide groups in the oxyanion hole.
• The peptide (amide) N-H groups that form this
  oxyanion hole and provide primary stabilization
  of the tetrahedral intermediates are from of
  Ser195 and Gly193.

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The oxyanion hole
The oxyanion hole of chymotrypsin stabilizes the
tetrahedral oxyanion intermediate seen in the
mechanism of Figure 14.21.
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Aspartic proteases play many roles in humans
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The Aspartic Proteases

• Pepsin, chymosin, cathepsin D, renin and HIV-1
  protease
• All involve two Asp residues at the active site.
• These two Asp residues work together as general
  acid-base catalysts.
• Most aspartic proteases have a tertiary structure
  consisting of two lobes (N-terminal and
  C-terminal) with approximate two-fold symmetry.
• HIV-1 protease is a homodimer.

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The Aspartic Proteases
Most aspartic proteases exhibit a two-lobed
structure. Each lobe contributes one catalytic
aspartate to the active site. HIV-1 protease is
a homodimeric enzyme, with each subunit
contributing a catalytic Asp residue.
Figure 14.22 Structures of (a) HIV-1 protease, a
dimer, and (b) pepsin, a monomer. Pepsins
N-terminal half is shown in red the C-terminal
half is shown in blue.
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The Aspartic Proteases
Figure 14.23 pH-rate profile for pepsin.
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The Aspartic Proteases
Figure 14.23 pH-rate profile of HIV-1 protease.
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Aspartic Protease Mechanism

• Aspartic proteases show one relatively low pKa,
  and one relatively high pKa.
• These pKa values as determined from the pH-rate
  plot for the enzyme, have traditionally been
  attributed to the two aspartate residues.
• However, molecular dynamics simulations now show
  that aspartic proteases employ low-barrier
  hydrogen bonds (LBHBs) in their mechanism.
• The predominant catalytic factor in aspartic
  proteases is general acid-base catalysis.

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A Mechanism for the Aspartic Proteases
Figure 14.24 Mechanism for the aspartic
proteases. LBHBs play a role in states E, ES,
ET, EQ, and EPQ.
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HIV-1 Protease

• A novel aspartic protease
• HIV-1 protease cleaves the polyprotein products
  of the HIV genome.
• This is a remarkable imitation of mammalian
  aspartic proteases.
• HIV-1 protease is a homodimer - more genetically
  economical for the virus.
• Active site is two-fold symmetric.

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Proteolytic cleavage pattern for the HIV genome
Figure 14.26 HIV mRNA provides the genetic
information for synthesis of a polyprotein.
Cleavage yields the active products.
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Protease Inhibitors Block the Active Site of
HIV-1 Protease
Figure 14.27 HIV-1 protease complexed with the
inhibitor Crixivan (red) made by Merck. The
flaps that cover the active site are green
the catalytic active site Asp residues are violet.
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Protease Inhibitors Give Life to AIDS Patients

• Protease inhibitors as AIDS drugs
• If the HIV-1 protease can be selectively
  inhibited, then new HIV particles cannot form.
• Several novel protease inhibitors are currently
  marketed as AIDS drugs.
• Many such inhibitors work in a culture dish.
• However, a successful drug must be able to kill
  the virus in a human subject without blocking
  other essential proteases in the body.

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End Chapter 14Mechanisms of Enzyme Action