Draw the structure of glutamic acid at pH 7'

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• Draw the structure of glutamic acid at pH 7.
• NH3
• HC-CH2-CH2-COO-
• COO-
• What are the pKas of the a-carboxyl and the
  g-carboxyl of Glu?
• ?-carboxyl pKa 3.7
• ?-carboxyl pKa 4.4
• They are slightly different. Why?
• Negative charge on the ?-carboxyl is stabilized
  by the positive charge on the ?-amino group.
• How much energy does it take to change the pKa of
  His from pKa 7 to pKa 5?
• Ka H A- /
  HA
• ?G -RTlnKeq -2.3RTlogKa
  2.3RTpKa
• ??G 2.3RT?pKa

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Lecture 1 questions

• What is the role of His64 in the mechanism of
  carbonic anhydrase.

polarization and orientation of reactive groups
His64
metal ion catalysis
N
H
?
? -
O
His
C
Zn2
His
HCO3-
O
O-
His
H
polarization and orientation of reactive groups
H
O
OH
O
C
C
Thr199
Glu106
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• Size, proximity orientation
• How are the catalytic groups oriented?
• Proximity, orientation and specificity in
  catalysis
• Binding energy and entropy.
• Role of transition state binding in catalysis
• Catalytic antibodies
• Molecular complexes
• Multienzyme complexes
• Signal transduction complexes
• Substrate Tunneling

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Proximity and orientation in enzyme catalysis
reactive end

• Binding energy is used to orient co-enzymes

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Proximity, orientation and specificity in enzyme
catalysis
Orientation of peptide CO
His
Asp
Ser
?-
.
.
.
.
CO
HN
?
H-O
N
HN
CO
O-
chymotrypsin
Orientation of catalytic triad
Specificity pocket
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Proximity, orientation and specificity in enzyme
catalysis
Orientation of peptide CO
His
Asp
Ser
?-
.
.
.
.
CO
HN
?
H-O
N
HN
-
H3N
trypsin
Orientation of catalytic triad
Specificity pocket
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Bovine Trypsin
Trypsin Inhibitor
Surface complimentarity
Specificity pocket
9
Reaction mechanism of serine proteases
R
His 57
Asp 102
Ser 195
?-
.
.
CO
?
HN
Gly 193
.
.
H-O
N
HN
HN
pKa 6.8
catalytic base
R
Tetrahedral Intermediate I
R
?-
His 57
Asp 102
C-O-
HN
Ser-O

C-O -
HN
N - H
HN
O
catalytic acid
R
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Reaction mechanism of serine proteases
acyl intermediate
R
His 57
Asp 102
CO
Ser-O
HN
Gly 193
N
HN
H-N-H
R
R
His 57
Asp 102
CO
Ser-O
HN
Gly 193
N
HN
H
O
catalytic base
H
nucleophilic water
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Reaction mechanism of serine proteases
R
His 57
Asp 102
C-O-
Ser-O
HN
Gly 193

NH
HN
C-O -
O
O
catalytic acid
Tetrahedral Intermediate II
H
His 57
Asp 102
HN
Gly 193
Ser-OH
R
N
HN
CO
O
H
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Catalytic Mechanisms and Strategies What
chemical groups are involved in catalysis? How
are the catalytic groups oriented? (Size) What
drives catalysis? (Energy) How is molecular
motion involved in catalysis? (Time)
H2O
RO
H
O
HOCH2
..
HEW Lysozyme
O
HO
NHAc
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Catalytic mechanism of HEW Lysozyme
General acid catalysis
Glu35
Glu35
C
O
C
O
O
H
O
RO
R
O
HOCH2
..
Electrostatic stabilization
O
HO
NHAc
O
O
O
C
O
C
Asp52
Asp52
Glu35
C
O
O
H
O
H
Nucleophilic attack by water
Configuration at retained
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Solvent accessibility of Glu35 and Asp52 in HEW
Lysozyme (using PDBviewer - color residues by
acessibility)
Asp52
Glu35
Yellow, orange, green solvent accessible Blue
solvent inaccessible
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Catalytic mechanism of aspartic proteases
- NH - C - C - NH - C -
?
?-
O
?
?
H
H
?-
?-
pKa 1.2
pKa 4.7
C - O-
pH optimum 2-3 for pepsin
Asp
Asp

• Nucleophilic attack by polarized water
• Unusual electrostatic environment
• Substrate binds in cleft

Ferst, Chapter 16 Creighton, Chapter 9
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Binding of substrate displaces water
Asp residues are buried in deep cleft
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Enolase
Note Lys-NH2 pKa 10 in
solution Glu-COOH pKa 5 in solution
These are not in solution!
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Enolase
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Solvent accessibility of Mg and catalytic Glu
and Lys in enolase (1E9I)
Yellow, orange, green solvent accessible Blue
solvent inaccessible
Mg
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• Size, proximity orientation
• How are the catalytic groups oriented?
• Role of proximity, orientation and specificity
  in catalysis
• Binding energy and entropy.
• Role of transition state binding in catalysis
• Protein active sites catalytic groups and
  water
• Molecular complexes
• Multienzyme complexes
• Signal transduction complexes
• Substrate Tunneling

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transition state
kforward
kreverse
Energy
?Gf
ES complex
?Greleased
EP complex
Reaction Coordinate
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transition state
Decrease of entropy Destabilizing electrostatic
interactions Km gt S
E S
Increase of entropy Stabilizing electrostatic
hydrogen bonding van der Waals
interactions Km lt S
?Gf
EP complex
ES complex
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• Entropy changes upon substrate binding involve
• restriction in substrate degree of freedom
  (unfavorable)
• restriction in side chain motion (unfavorable)
• release of surface-bound water (favorable)

?G -25 - -50 cal/mol for every 1 Å2 of surface
area buried
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transition state
Decrease entropy of EP complex Destabilizing
electrostatic interactions
?Gf
products
ES complex
Increase entropy of products
EP complex
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• Size, proximity orientation
• How are the catalytic groups oriented?
• Role of proximity, orientation and specificity
  in catalysis
• Binding energy and entropy.
• Role of transition state binding in catalysis
• Catalytic antibodies
• Molecular complexes
• Multienzyme complexes
• Signal transduction complexes
• Substrate Tunneling

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transition state
kforward
kreverse
Energy
ES complex
?Gf
EP complex
Reaction Coordinate
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transition state
substrate
products
?Gf
Reaction Coordinate
28
transition state
ES complex
ES complex
?Gf
EP complex
Enzyme binding site is complementary to the
structure of the substrate.
Reaction Coordinate
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transition state
ES complex
transition state
ES complex
?Gf
EP complex
Enzyme binding site is complementary to the
transition state structure of the substrate.
Reaction Coordinate
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Catalytic mechanism of HEW Lysozyme
General acid catalysis
Glu35
Glu35
C
O
C
O
O
H
O
RO
R
O
HOCH2
..
Electrostatic stabilization of transition state
O
HO
NHAc
O
O
O
C
O
C
Asp52
Asp52
Glu35
C
O
O
H
O
H
Nucleophilic attack by water
Configuration at retained
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antigen
Catalytic antibodies
Synthesize chemical analog to transition state
structure
Raise antibodies to transition state analog
antibody
Add normal substrate to antibody and see if it
will catalyze the reaction
antibody
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• Size, proximity orientation
• How are the catalytic groups oriented?
• Role of proximity, orientation and specificity
  in catalysis
• Binding energy and entropy.
• Role of transition state binding in catalysis
• Catalytic antibodies
• Molecular complexes
• Multienzyme complexes
• Signal transduction complexes
• Substrate Tunneling

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• Advantages of Multisubunit Complexes
• Catalytic enhancement. Reduction of diffusion
  time of an intermediate from one enzyme to the
  next.
• Substrate channeling. Control over the
  biosynthetic route an intermediate should follow,
  by directing it to a specified enzyme rather than
  allowing competition.
• Sequestration of reactive intermediates.
  Protection of chemically unstable species from
  aqueous solution.
• Servicing. Where one subunit can pass a
  reagent to many different other subunits.
• Coordinate regulation and expression.

Fersht, Chapter 1
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Pyruvate dehydrogenase complex -no crystal
structure of full complex

• 60 proteins 24 E1, 24 E2, 12 E3
• MW 4600 kDa
• Dimer of E1 at on each edge of cube
• 12 x 2 24 subunits
• Trimer of E2 at each corner of cube
• 8 x 3 24 subunits
• Dimer of E3 on each face of cube
• 6 x 2 12 subunits
• Forms acetyl CoA from pyruvate
• Involves NADH, lipoic acid, FAD,
• TPP, acetyl CoA

E1
E3
E2
300 Å
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Pyruvate Dehydrogenase Complex
CO2
H3C
pyruvate
lipoic acid
O
C
S-CH2
H3C
S-CH2
COO-
O
S
C
R
S
R
S-CH
S-CH
N
N
R
R
TPP
E2
E1
E2
E1
HS-CH2-CoA
HS-CH2-CoA
acetyl CoA
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Pyruvate Dehydrogenase Complex
H3CCS-CH2
HS-CH2
S
R
S
S-CH
R
H
HS-CH
N
N
R
R
E2
E1
E2
E1
HS-CH2-CoA
CH3-C S-CH2-CoA
Once acetylated, the long liposyl arm can
rapidly exchange/pass the acetyl group to the
other 23 subunits.
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Pyruvate Dehydrogenase Complex
H
O
O
H3C
HS-CH2
H
H3C
N
S-CH2
H
N
N
N
HS-CH
N
O
S-CH
N
H3C
N
O
N
H3C
H
H
O
H
O
C
C
NH2
C
C
NH2
E3
E2
E3
E2


O
N
O
N
CH3-C S-CH2-CoA
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Pyruvate Dehydrogenase Complex
O
O
H3C
H
N
H3C
S-CH2
H
N
N
S-CH2
N
S-CH
N
O
N
H3C
S-CH
N
O
N
H3C
H
H
O
C
C
NH2
E3
E2
E3
E2

O
N
NADH
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Signal tranduction complexes form at membrane
surface
Protein-protein and protein-membrane
interactions involve SH2 domains SH3 domains PH
domains etc. and Phosphorylation
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• Size, proximity orientation
• How are the catalytic groups oriented?
• Role of proximity, orientation and specificity
  in catalysis
• Binding energy and entropy.
• Role of transition state binding in catalysis
• Protein active sites catalytic groups and
  water
• Molecular complexes
• Multienzyme complexes
• Signal transduction complexes
• Substrate Tunneling

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Carbamoyl phosphate synthetase II in pyrimidine
biosynthesis
HCO3- glutamine 2ATP H20
O
O
H2N C O P O- 2ADP Pi glutamate
O-
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Carbamoyl phosphate synthetase II
Glutamine binds
Releases NH4
Diffuses down channel (100 Å long)
Combines with bicarbonate in a reaction requiring
ATP
Phosphorylated to form carbamoyl phosphate
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H released outside

• Bacteriorhodopsin
• Membrane protein
• Proton pump
• Light activated
• Internal H channel

Retinal isomerization transfer proton from lower
to upper channel.
H enters from cytoplasm
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Lecture 2 questions
1. A binding constant of 10 micromolar is good
but not great. How many well placed hydrogen
bonds would it take to increase this binding
constant to 1 nanomolar (a binding constant that
reflects a tight interaction)?