PHYSICAL ORGANIC CHEMISTRY

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PHYSICAL ORGANIC CHEMISTRY

• II. Thermodynamics of host-guest formation
  entropy-enthalpy compensation, enzyme catalysis,
• design of enzymes.
• K. N. Houk
• Department of Chemistry and Biochemistry

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Survey of Host-Guest Equilibrium Binding Constants
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Equilibrium constant measurements direct and
indirect
H G HG indirect measurement of KD from
IC50 HG G HG G IC50 concentration to
displace 50 of G from HG KD IC50/(1
G0)/KD) G0 initial concentration of G
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?-, ?-, and ?-Cyclodextrins
Ka 102.5?1.1 M-1 DG -3.5?1.4 kcal/mol
1257 examples
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How do enzymes compare? Enzyme-substrate Enzym
e-transition state Enzyme-inhibitor
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I. D. Kuntz, K. Chen, K. A. Sharp, and P. A.
Kollman, PNAS, USA, 1999, 96, 9997.
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Comparing Bound Surface Area with Binding Affinity
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Enthalpy-Entropy Compensation
DH0 TcompensationDS0
Tcompensation is often referred to as ?

• Physically meaning relationship resulting from
  the fact that
• more favorable (more negative) enthalpy of
  binding is
• accompanied by less favorable (more negative)
  entropy.
• and or
• Results from small experimental errors that
• produce a statistically insignificant
  relationship.

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Gilli et al. propose that Enthalpy-Entropy
Compensation limits KD to 10pm (10-11, 15
kcal/mol or 65 kJ/mol)
P. Gilli et al., J. Phys. Chem. 1994, 98, 1515.
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But Enthalpy-Entropy Compensation can result
from random errors
A. Cornish-Bowden, Enthalpy-entropy compensation
a phantom phenomenon
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• Interactions Determining Host-Guest Complex
  Stabilities
• Hostsolvated Guestsolvated ------gt
  Host-Guest Complexsolvated
• Bohms LUDI
• Electrostatic interactions ionic (up to
  -2kcal/mol)
• Hydrogen-bonding HB (up to -1.2kcal/mol)
• Solvation and the hydrophobic effect lipophilic
  (-0.04kcal/mol/A2)
• Guest reorganization penalty conf (0.3 per
  rotatable bond)

Scoring functions using these additive energy
functions, have standard deviations
from experiment of about 1 kcal/mol for single
receptors and 2 kcal/mol more generally.
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Hydrogen-bonding
Strong (short-strong or low barrier)
F---H---F- can be 40 kcal/mol in gas phase, but
disappear in H2O Coulombs Law vacuum
e?? 1 DE 332 q1q2/r12e CCl4, PhH
2 (in kcal/mol) DMSO 47 H2O 80 Medium N
-H---OC about 5 kcal/mol in the gas phase, but
only about 1 kcal/mol relative to H2O Weak
C-H---OC about 1 kcal/mol in the gas phase.
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The Role of Solvent-Accessible Surface Areas
The relationship between binding affinity and
DSASA is evidence for the
The Hydrophobic Effect
The binding free energy that causes nonpolar
molecules (or regions of molecules) to bind
relative to water. The hydrophobic free energy
is found to be related to the solvent
accessible surface area that is buried (removed
from access to water) upon binding.
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Comparing Bound Surface Area with Binding Affinity
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Average Binding Affinity is determined by ligand
and host cavity size, which is related to the
change in solvent accessible surface area bound
upon host-guest complex formation. Hydrophobic
effects (or solvophobic effects) have the major
influence on average binding energies for a given
class of hosts. Selectivity for binding with
respect to a given host is determined by
remaining factors, such as electrostatic (especial
ly hydrogen bonding), desolvation and
guest restriction energies.
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Drug development - identifying inhibitors or
activatorsof enzymes and receptor proteins that
bind selectively with nanomolar to picomolar
affinity ?DG 13-17 kcal/mol) (or 55-71 kJ/mol).
But there is the matter of ADMET Absorption Distr
ibution Metabolism Excretion Toxicity
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A Wolfenden Plot
log k

B. G. Miller and R. Wolfenden, Annu. Rev.
Biochem. 2002, 71847-85
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What is the origin of the enormous acceleration
of rates of reactions by enzymes?
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Michaelis-Menten kinetics
Vmax
S
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Quantitative measures of enzyme
catalysis (diagram represents DG corresponding to
Michaelis rates and equilibria)
kcat/kun rate enhancement
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The specificity of the physiological activity of
substances is determined by the size and shape of
molecules, rather than primarily by their
chemical properties, and that the size and shape
find expression by determining the extent to
which these regions of the two molecules are
complementary in structure. The enzyme is
closely complementary in structure to the
activated complex for the reaction catalyzed by
the enzyme. Pauling, L. Chemical
Engineering News 1946, 24, 13751377.
enzymes are molecules that are complementary in
structure to the activated complexes of the
reactions that they catalyze, , rather than
entering into reactions. Pauling, L. Nature
1948, 161, 707709.
The enzyme has a configuration complementary to
the activated complex, and accordingly has the
strongest power of attraction for the activated
complex. Pauling, L. American Scientist
1948, 36, 5158.
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Legacy of the Pauling Paradigm

• Drug Design

HIV-1 reverse transcriptase
Nevirapin
Cat. Ab. 1E9
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Extraordinary binding of transition states
(or lowering of the free energy of activation) by
enzymes has been attributed to

• Various non-covalent physical interactions such
  as
• Preorganization and electrostatic stabilization
  oriented dipoles
• complementary to the TS (Warshel et al.)
• Ground state destabilization and desolvation
  (Lienhard, Dewar, Gao et al.)
• "Circe effect - attractive binding destabilizes
  substrate (Jencks)
• Entropy trap - binding of two substrates
  overcomes -DS (Westheimer)
• Approximation, Proximity, Propinquity,
  Togetherness, etc. (Koshland,
• Bruice, Jencks, et al.)
• Orbital steering (Koshland)
• NACs - stabilization of near attack conformations
  (Bruice)
• Spatiotemporal hypothesis (Menger)
• Dynamic coupling of protein fluctuations with
  motions in the TS (Brooks,
• Benkovic et al.)
• Dynamic enhancement of tunneling (Klinman et al.)
• Induced fit (Koshland)
• Noncovalent cooperativity and enhanced enzyme
  packing (Williams)
• Factors that involve covalent or partially
  covalent chemical interactions and
• a change in the mechanism of the reaction

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Extraordinary binding of transition states
(or lowering of the free energy of activation) by
enzymes has been attributed to

• Preorganization and electrostatic stabilization
  oriented dipoles
• complementary to the TS (Warshel et al.)
• Various non-covalent physical interactions such
  as
• Ground state destabilization and desolvation
  (Lienhard, Dewar, Gao et al.)
• "Circe effect - attractive binding destabilizes
  substrate (Jencks)
• Entropy trap - binding of two substrates
  overcomes -DS (Westheimer)
• Approximation, Proximity, Propinquity,
  Togetherness, etc. (Koshland,
• Bruice, Jencks, et al.)
• Orbital steering (Koshland)
• NACs - stabilization of near attack conformations
  (Bruice)
• Spatiotemporal hypothesis (Menger)
• Dynamic coupling of protein fluctuations with
  motions in the TS (Brooks,
• Benkovic et al.)
• Dynamic enhancement of tunneling (Klinman et al.)
• Induced fit (Koshland)
• Noncovalent cooperativity and enhanced enzyme
  packing (Williams)
• Factors that involve covalent or partially
  covalent chemical interactions and
• a change in the mechanism of the reaction

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Association constants for antibodies and enzymes
Covalent and partially covalent chemical catalysis
Non-covalent physical interactions
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Binding energy vs. number of heavy (non-H)
atoms in inhibitor
D. Kuntz, K. Chen, K. A. Sharp, and P. A.
Kollman, PNAS, USA, 1999, 96, 9997 see also P.
J. Hajduk, J. Med. Chem. 2006, 49, 6972-6076.
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Extraordinary Binding of Transition States by
Enzymes
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UPDATING THE PAULING PARADIGM

Specificity is determined by non-covalent
molecular recognition (H-bond, electrostatic,
hydrophobic and van der Waals interactions)
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Houk - 49
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A Wolfenden Plot
How do these enzymes work?
log k

B. G. Miller and R. Wolfenden, Annu. Rev.
Biochem. 2002, 71847-85
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A survey of literature on mechanisms of
Wolfendens enzymes shows
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A Proficiency Plot based on estimations of kun
for other enzymes
1075 enzymes
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Why the controversy? Semantic issues?
Use of difference reference state?
catalytic groups (change in mechanism)
Reaction in water by same mechanism as enzyme
pre-organization (environmental changes
in active site)
Reaction in water
Reaction in enzyme
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Can enzymes for non-natural reactions be designed
and synthesized?
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Major Progress in Inverse Protein Folding

• Approximate energy functions and minimization
  algorithms for identifying amino acid sequences
  compatible with a target tertiary structure
• A milestone was reached with the design and
  successful experimental proof of structure of a
  93-residue ?/? protein called Top7 by David
  Bakers group

Kuhlman, B. Dantas, G. Ireton, G.C. Varani,
G. Stoddard, B.L. Baker, D. Science, 2003, 302,
1364-1368
The next great challenge of protein design to
predict and synthesize a functional protein
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Designing New Enzyme Catalysts
Probability of generation of a functional
protein ? 1/1077
(Axe D. D. J. Mol. Biol. 2004, 341, 1295)

• Narrowing the sequence selection to a small area
  of an existing protein or enzyme scaffold could
  render enzyme design more feasible.

• The retrofitting strategy can produce hybrid
  enzymes, a term coined by Benkovic et al. for
  proteins containing elements from more than one
  functional protein.

Nixon, A. E. Ostermeier, M. Benkovic, S. J.
Trends Biotechnol. 1998, 16, 258-264.
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Designing Active Site Architecture into a Binding
Protein
H. W. Hellinga
TIM
RBP
kcat/kuncat ? 105-6
Dwyer, M. A. Looger, L.L Hellinga, H. W.
Science, 2004, 304, 196-197
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Designing Esterase Functionality into Thioredoxin
S. L. Mayo
Thioredoxin scaffold
Designed Esterase
kcat/kuncat ? 180
Bolon, D.N. Mayo, S.L. Proc. Natl. Acad. Sci.
USA 2001, 98, 14274-14279
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Protein Design Processes

• Inside-out protocol to design novel enzyme
  catalysts
• Baker - Houk collaboration

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Wang, K. C. Lai, C.-F. Lee, J.-Y. J. Taiwan
Pharm. Assoc. 1979, 31, 63-70.

• Five designs using three different scaffolds
  expressed and tested
• 6-14 mutations from the original scaffolds
• No aldolase activity (not even at 11
  enzymesubstrate, or different pH ranging from
  5.0 to 11.0)
• Slow enamine formation for one of the designs

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Does it fold properly? Is the designed active
site realized? Does the designed active site
function?
The designed aldolase - based on the TIM Barrel
from an imidazoleglycerol phosphate synthase