The Organic Chemistry of Enzyme-Catalyzed Reactions Revised Edition

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The Organic Chemistry of Enzyme-Catalyzed
ReactionsRevised Edition

• Professor Richard B. Silverman
• Department of Chemistry
• Department of Biochemistry, Molecular Biology,
  and Cell Biology
• Northwestern University

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The Organic Chemistry of Enzyme-Catalyzed
Reactions Chapter 1 Enzymes as Catalysts
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For published data regarding any enzyme
see http//www.brenda-enzymes.info/
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What are enzymes, and how do they work?

• First isolation of an enzyme in 1833
• Ethanol added to aqueous extract of malt
• Yielded heat-labile precipitate that was utilized
  to hydrolyze starch to soluble sugar precipitate
  now known as amylase
• 1878 - Kühne coined term enzyme - means in
  yeast
• 1898 - Duclaux proposed all enzymes should have
  suffix ase

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• Enzymes - natural proteins that catalyze chemical
  reactions
• First enzyme recognized as protein was jack bean
  urease
• Crystallized in 1926
• Took 70 more years (1995), though, to obtain its
  crystal structure

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• Enzymes have molecular weights of several
  thousand to several million, yet catalyze
  transformations on molecules as small as carbon
  dioxide and nitrogen
• Function by lowering transition-state energies
  and energetic intermediates and by raising the
  ground-state energy
• Many different hypotheses proposed for how
  enzymes catalyze reactions
• Common link of hypotheses enzyme-catalyzed
  reaction always initiated by the formation of an
  enzyme-substrate (or E?S) complex in a small
  cavity called the active site

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• 1894 - Lock-and-key hypothesis - Fischer proposed
  enzyme is the lock into which the substrate (the
  key) fits
• Does not rationalize certain observed phenomena
• Compounds having less bulky substituents often
  fail to be substrates
• Some compounds with more bulky substituents bind
  more tightly
• Some enzymes that catalyze reactions between two
  substrates do not bind one substrate until the
  other one is bound

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• 1958 - Induced-fit hypothesis proposed by
  Koshland
• When a substrate begins to bind to an enzyme,
  interactions induce a conformational change in
  the enzyme
• Results in a change of the enzyme from a low
  catalytic form to a high catalytic form
• Induced-fit hypothesis requires a flexible active
  site

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• Concept of flexible active site stated earlier by
  Pauling (1946)
• Hypothesized that an enzyme is a flexible
  template that is most complementary to substrates
  at the transition state rather than at the ground
  state
• Therefore, the substrate does not bind most
  effectively in the E?S complex
• As reaction proceeds, enzyme conforms better to
  the transition-state structure
• Transition-state stabilization results in rate
  enhancement

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• Only a dozen or so amino acid residues may make
  up the active site
• Only two or three may be involved directly in
  substrate binding and/or catalysis

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• Why is it necessary for enzymes to be so large?
• Most effective binding of substrate results from
  close packing of atoms within protein
• Remainder of enzyme outside active site is
  required to maintain integrity of the active site
• May serve to channel the substrate into the
  active site
• Active site aligns the orbitals of substrates and
  catalytic groups on the enzyme optimally for
  conversion to the transition-state structure--
  called orbital steering

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• Enzyme catalysis characterized by two features
  specificity and rate acceleration
• Active site contains amino acid residues and
  cofactors that are responsible for the above
  features
• Cofactor, also called a coenzyme, is an organic
  molecule or metal ion that is essential for the
  catalytic action

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Specificity of Enzyme-Catalyzed Reactions

• Two types of specificity (1) Specificity of
  binding and (2) specificity of reaction
• Specificity of Binding
• Enzyme catalysis is initiated by interaction
  between enzyme and substrate (E?S complex)
• k1, also referred to as kon, is rate constant for
  formation of the E?S complex
• k-1, also referred to as koff, is rate constant
  for breakdown of the complex
• Stability of E?S complex is related to affinity
  of the substrate for the enzyme as measured by
  Ks, dissociation constant for the E?S complex

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Generalized enzyme-catalyzed reaction
kon
Michaelis complex
koff
Scheme 1.1
When k2 ltlt k-1, k2 called kcat (turnover
number) Ks called Km (Michaelis-Menten constant)
kcat represents the maximum number of substrate
molecules converted to product molecules per
active site per unit of time called turnover
number
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• Km is the concentration of substrate that
  produces half the maximum rate
• Km is a dissociation constant, so the smaller the
  Km the stronger the interaction between E and S
• kcat/Km is the specificity constant - used to
  rank an enzyme according to how good it is with
  different substrates

kcat
Upper limit for is rate of diffusion (109
M-1s-1)
Km
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• How does an enzyme release product so
  efficiently given that the enzyme binds the
  transition state structure about 1012 times more
  tightly than it binds the substrate or products?
• After bond breaking (or making) at transition
  state, interactions that match in the
  transition-state stabilizing complex are no
  longer present.
• Therefore products are poorly bound, resulting in
  expulsion.
• As bonds are broken/made, changes in electronic
  distribution can occur, generating a repulsive
  interaction, leading to expulsion of products

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E S complex
Figure 1.1
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?Gº -RTlnKeq
If Keq 0.01, ?Gº of -5.5 kcal/mol needed to
shift Keq to 100
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Examples of ionic, ion-dipole, and dipole-dipole
interactions. The wavy line represents the enzyme
active site
Specific Forces Involved in ES Complex Formation
Figure 1.2
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Hydrogen bonding in the secondary structure of
proteins ?-helix and ?-sheet.
H-bonds
H-bonds A type of dipole-dipole interaction
between X-H and Y (N, O)
Figure 1.3
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Charge Transfer Complexes

• When a molecule (or group) that is a good
  electron donor comes into contact with a molecule
  (or group) that is a good electron acceptor,
  donor may transfer some of its charge to the
  acceptor

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Hydrophobic Interactions

• When two nonpolar groups, each surrounded by
  water molecules, approach each other, the water
  molecules become disordered in an attempt to
  associate with the water molecules of the
  approaching group
• Increases entropy, resulting in decrease in the
  free energy (?G? ?H?-T?S?)

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van der Waals Forces

• Atoms have a temporary nonsymmetrical
  distribution of electron density resulting in
  generation of a temporary dipole
• Temporary dipoles of one molecule induce opposite
  dipoles in the approaching molecule

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Binding Specificity

• Can be absolute or can be very broad
• Specificity of racemates may involve ES complex
  formation with only one enantiomer or ES complex
  formation with both enantiomers, but only one is
  converted to product
• Enzymes accomplish this because they are chiral
  molecules (mammalian enzymes consist of only
  L-amino acids)

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Resolution of a racemic mixture
Binding specificity of enantiomers
diastereomers
Scheme 1.2
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• Binding energy for ES complex formation with one
  enantiomer may be much higher than that with the
  other enantiomer
• Both ES complexes may form, but only one ES
  complex may lead to product formation
• Enantiomer that does not turn over is said to
  undergo nonproductive binding

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Basis for enantioselectivity in enzymes
Steric hindrance to binding of enantiomers
Leu
S
R
Figure 1.4
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Reaction Specificity

• Unlike reactions in solution, enzymes can show
  specificity for chemically identical protons

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Enzyme specificity for chemically identical
protons. R and R? on the enzyme are groups that
interact specifically with R and R?,
respectively, on the substrate.
Figure 1.5
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Rate Acceleration

• An enzyme has numerous opportunities to invoke
  catalysis
• Stabilization of the transition state
• Destabilization of the ES complex
• Destabilization of intermediates
• Because of these opportunities, multiple steps
  may be involved

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Effect of (A) a chemical catalyst and (B) an
enzyme on activation energy
Figure 1.6
1010-1014 fold typically
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Enzyme catalysis does not alter the equilibrium
of a reversible reaction it accelerates
attainment of the equilibrium
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Mechanisms of Enzyme Catalysis Approximation

• Rate enhancement by proximity
• Enzyme serves as a template to bind the
  substrates
• Reaction of enzyme-bound substrates becomes first
  order
• Equivalent to increasing the concentration of the
  reacting groups
• Exemplified with nonenzymatic model studies

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Second-order reaction of acetate with aryl acetate
Scheme 1.3
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Table 1.3. Effect of Approximation on Reaction
Rates
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Nucleophilic catalysis
Covalent Catalysis
anchimeric assistance
Scheme 1.4
Most common Cys (SH) Ser (OH) His
(imidazole) Lys (NH2) Asp/Glu (COO-)
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Anchimeric assistance by a neighboring group
Scheme 1.5
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Early evidence to support covalent catalysis
Model Reaction for Covalent Catalysis
Scheme 1.6
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General Acid/Base Catalysis
This is important for any reaction in which
proton transfer occurs
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The catalytic triad of ?-chymotrypsin. The
distances are as follows d1 2.82 Å d2 2.61
Å d3 2.76 Å.
Figure 1.7
catalytic triad
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Charge relay system for activation of an
active-site serine residue in ?-chymotrypsin
Scheme 1.7
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• pKa values of amino acid side-chain groups within
  the active site of enzymes can be quite different
  from those in solution
• Partly result of low polarity inside of proteins
• Molecular dynamics simulations show
  interiors of these proteins have dielectric
  constants of about 2-3 (dielectric constant for
  benzene or dioxane)
• If a carboxylic acid is in a nonpolar region, pKa
  will rise
• Glutamate-35 in the lysozyme-glycolchitin complex
  has a pKa of 8.2 pKa in solution is 4.5
• If the carboxylate ion forms salt bridge, it is
  stabilized and has a lower pKa

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• Basic group in a nonpolar environment has a lower
  pKa
• pKa of a base will fall if adjacent to other
  bases
• Active-site lysine in acetoacetate decarboxylase
  has a pKa of 5.9 (pKa in solution is 10.5)

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• Two kinds of acid/base catalysis
• Specific acid or specific base catalysis -
  catalysis by a hydronium (H3O) or hydroxide
  (HO-) ion, and is determined only by the pH
• General acid/base catalysis - reaction rate
  increases with increasing buffer concentration at
  a constant pH and ionic strength

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Effect of the buffer concentration on (A)
specific acid/base catalysis and (B) general
acid/base catalysis
Specific acid/base catalysis
General acid/base catalysis
Figure 1.8
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Hydrolysis of ethyl acetate
Specific Acid-Base Catalysis
Scheme 1.8
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Alkaline hydrolysis of ethyl acetate
Scheme 1.9
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Acid hydrolysis of ethyl acetate
Scheme 1.10
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Simultaneous acid and base enzyme catalysis
Enzymes can utilize acid and base catalysis
simultaneously
acid catalysis
base catalysis
Scheme 1.11
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Simultaneous acid/base catalysis is the reason
for how enzymes are capable of deprotonating weak
carbon acids
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Simultaneous acid and base enzyme catalysis in
the enolization of mandelic acid
Scheme 1.12
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• Low-barrier hydrogen bonds - short (lt 2.5Å), very
  strong hydrogen bonds
• Stabilization of the enolic intermediate occurs
  via low-barrier hydrogen bonds

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Simultaneous acid and base enzyme catalysis in
the 1,4-elimination of ?-substituted (A)
aldehydes, ketones, thioesters and (B) carboxylic
acids
low-barrier H-bond
One-base mechanism syn-elimination
strong acid
weak base
stronger acid needed
low-barrier H-bond
Two-base mechanism anti-elimination
strong base
weak acid
Scheme 1.13
carboxylic acids
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Base catalyzed 1,4-elimination of ?-substituted
carbonyl compounds via an enolate intermediate
(ElcB mechanism)
ElcB mechanism - not relevant
Needs acid or metal catalysis
Scheme 1.14
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Electrostatic enzyme catalysis in enolization
Alternative to Low-Barrier Hydrogen Bond
Scheme 1.15
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Electrostatic stabilization of the transition
state
Electrostatic Catalysis
oxyanion hole
Scheme 1.16
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Desolvation
The removal of water molecules at the active site
on substrate binding

• Exposes substrate to lower dielectric
  constant environment
• Exposes water-bonded charged groups for
  electrostatic catalysis
• Destabilizes the ground state

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Alkaline hydrolysis of phosphodiesters
Strain Energy
Scheme 1.17
k1.7
108
k1.8
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Induced Fit Hypothesisputting strain energy into
the substrate
Figure 1.9
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Energetic Effect of Enzyme Catalysis
Figure 1.10
Importance of ground state destabilization
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Mechanisms of Enzyme Catalysis - porphobilinogen
synthase