The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 13
Rearrangements
Rearrangements
Pericyclic Reactions - concerted reactions in
which bonding changes occur via reorganization
of electrons within a loop of interacting orbitals
Sigmatropic Rearrangements
[3,3] sigmatropic rearrangement
General form of the Claisen rearrangement
O
O
H
Scheme 13.1
Chorismate Mutase-catalyzed Conversion
of Chorismate to Prephenate
Scheme 13.2
7
O
9
COO1
9
2
6
CH2
3
5
4
HO
CH2 C COO-
-OOC
O 8 COO7
8
1
6
2
5
3
4
HO
13.1
13.2
chorismate
prephenate
A step in the biosynthesis of Tyr
and Phe in bacteria, fungi, plants
Conformation of Chorismate in Solution
Required conformer for Claisen rearrangement
(10-40% observed in solution from NMR spectrum)
-O
2C
O
COO-
OH
13.6
chair-like TS‡
Evidence for Chairlike Transition State
Stereochemical outcome if chorismate mutase
proceeds via chair and boat transition states,
respectively, during reaction with (Z)-[9-3H]chorismate
-OOC
H
3H
-OOC
O
COO-
A
COOchair
OH
13.8
Z-13.7
O
B
pro-R
H
COOCOO-
3H
COO-
H
O
COO-
boat
OH
OH
Scheme 13.3
3H
O
OH
3H
pro-S
H
Z-13.7
13.9
To Determine the Position of the 3H
Chemoenzymatic degradation of the prephenate formed
from the chorismate mutase-catalyzed conversion of (Z)-[93H]chorismate to determine the position of the tritium
Scheme 13.4
COO-
COOHS
HR
-OOC
HS
COOO
HR
pH < 6
- CO2 , -H2O
HS
O
phenylpyruvate
tautomerase
-HR+
OH
Z-[9- 3H]chorismate
E-[9- 3H]chorismate
20% 3H release
67% 3H release
Therefore, chair TS‡
OH
COO-
Five Hypothetical
Stepwise Mechanisms for
the Reaction Catalyzed
by Chorismate Mutase
COO-
(1)
+
4
B+ H
COO-
O
rearrangement
OH
-OOC
COOO
H2O
+
-OOC
Figure 13.1
COO-
O
(2)
COO-
-OOC
+O
+
prephenate
COO-
O
COO-
COOB:
B+ H
O
H O
COO-
COO-
(3)
prephenate
+
OH
OH
COOO
COO-O
COO-
COO-
(4)
prephenate
H O
OH
X H
X
H B+
:B
2° inverse isotope effect on
C-4 (sp2  sp3); therefore
not 1-3 (sp3  sp2)
COOO
COO-O
COO-
(5)
COOprephenate
B:
H O
B+
O
H
Both are substrates
COO-
COO-
O
COOO
COO-
OCH3
13.10
13.11
mechanism 5 excluded
mechanisms 1, 2, 5 excluded
16 mutants made to show neither general acid-base catalysis
(mechanisms 1-3, 5) nor nucleophilic catalysis (mechanism
4) is important
Conclusion: pericyclic
Function of the enzyme is to stabilize the chair transition
state geometry
Oxy-Cope Rearrangement
General form of Cope (A) and oxy-Cope (B) reactions
Cope
A
OH
OH
O
oxy-Cope
B
Scheme 13.5
Neither observed yet by an enzyme,
but a catalytic antibody has been raised
Oxy-Cope Rearrangement
Catalyzed by an Antibody
COOH
‡
COOH
COOH
bond
rotation
OH
OH
HO
13.14
13.13
COOH
COOH
COOH
bond
rotation
O
13.15
O
Scheme 13.6
OH
O
N
H
O
OH
13.12
hapten to raise the antibody
NH2
[2,3] Sigmatropic Rearrangement Catalyzed
by Cyclohexanone Oxygenase
O
Ph
Se
13.16
enzyme
NADPH
O2
Se
Ph
[2,3] sigmatropic
rearrangement
PhSe
O
H
•
•O
O
H
PhSe•
PhSe
H
•
13.17
Scheme 13.7
[4+2] Cycloaddition (Diels-Alder) Reaction
R
‡
H
R
R
H
R'
H
R'
H R'
H
R"
R"
R"
H
13.18
boat like TS‡
Scheme 13.9
(d,l)
An Intramolecular Diels-Alder Reaction
Catalyzed by Alternaria solani
OCH3
OHC
OCH3
OHC
O
O
O
O
exo
endo
OHC
OCH3
O
OHC
H
O
OCH3
O
H
O
H3 C
H
H3 C
H
Scheme 13.10
13.19a
enzymatic
exo : endo is 53 : 47
13.19b
solanopyrones
in aqueous solution exo : endo
is 3 : 97 (nonenzymatic)
An Antibody-Catalyzed Diels-Alder
Reaction
H
‡
O
N
NHAc
-OOC
O
NH
COO-
O
O
Scheme 13.11
O
H
NH
H
O
N
O
NHAc
N
O
+
O
NHAc
H
O
O
NH
O
COO-
Hapten used
O
-OOC
H
O
NH
H
O
13.20
N
O
NHAc
This hapten gives an
antibody that makes
only endo product
HN
O
CONMe2
O
This hapten gives an
antibody that makes
only exo product
CONMe2
HN
O
O
O
O
13.23
N
O
O
13.24
O
N
O
Rearrangements via a Carbenium Ion
An acid-catalyzed acyloin-type rearrangement
acid-catalyzed
+
H2O H
:O:
R
C
OH
OH
C
R'
R"
13.31
acyloins
Scheme 13.14
R
C
: OH
C
OH
R'
R"
[1,2] alkyl
migration
R
C
R"
OH2
+
O H
C
R'
R
OH
O
C
C
R"
R'
Reactions Catalyzed by Acetohydroxy
Acid Isomeroreductase
O
CH3 C
HO
OH
C
COO-
+
NADP3H
CH3
C
OH
C
COO-
+ NADP+
13.33
13.32
CH3 C
C
14CH 3H
3
14CH
3
O
OH
COO-
+
14CH CH
2
3
13.34
Scheme 13.15
NADP3H
CH3
OH
OH
C
C3H COO-
14CH CH
2
3
13.35
+ NADP+
OH O
CH3 C
C COO-
CH3
13.36
substrate
Kinetically-competent intermediate
Proposed Acyloin-type Mechanism for
Acetohydroxy Acid Isomeroreductase
Scheme 13.16
concerted
B
H
O
O
C
C
CH3
:B
H
COO-
CH3
C
R
+
OH OH
: O : OH
C
COO-
CH3
C
C
B:
C
COO-
CH3
C
R
R
NADPH + H+
NADP+
OH OH
CH3
C
C
R
H
C
+
COO-
C
COO-
R
H
HO
OH O
C
CH3
R
R
CH3
OH OH
COO-
O+
C
OH OH
COO-
OH OH
CH3
intermediate
+
B H
C
COO-
R
stepwise
CH3
C
NADP+
NADPH + H+
OH O
CH3
C
R
C
+
COO-
C
C
R
H
COO-
Cyclizations
Sterol biosynthesis
Conversion of squalene to lanosterol
H
20
12
10
1
18O
2
2
NADPH
3
H18O
11
9
14
10
4
5
17
13
8
15
7
H
16
cholesterol
6
13.38
13.37
lanosterol
squalene
squalene
2,3-epoxidase
NADPH
O2, flavin,
nonheme Fe2+
2,3oxidosqualenelanosterol cyclase
Scheme 13.17
O
Initial Mechanism Proposed for
2,3-Oxidosqualene-lanosterol Cyclase
2,3-oxidosqualene-lanosterol cyclase
not
isolated
B:
B+ H
:O
Me
2 Me
1
HO
X
squalene
2,3-epoxidase
Me
9
H
13 17
Me
14
8
H
anti-Markovnikov (to
get 6-membered ring)
20
Me
X
H 17 Me
13.40
protosterol
Me
Me
Me
HO
Scheme 13.18
Me
H
3
13.39
squalene
Me
H
Me
Me
Me
H
H
7 stereogenic
centers
(128 possible
isomers)
Me
13.38
Isotope labeling shows the 4 migrations are intramolecular
Covalent catalysis proposed to control stereochemistry
lanosterol
only isomer
formed
Me
Evidence for 17 Configuration
Use of 20-oxa-2,3-oxidosqualene to determine the
stereochemistry at C-17 of lanosterol from the reaction
catalyzed by 2,3-oxidosqualene-lanosterol cyclase
O instead of CH2
B+ H
O
H
Me
Me
O
Me
Me
O
HO
17
Me
H
13.41
Me
H
H
13.42
Scheme 13.19
no covalent
catalysis needed
Me
Me
H
Me
Me
O
HO
17
Me
H
Me
13.43
isolated
H
H
17
Further Support for Structure of Protosterol
Use of (20E)-20,21-dehydro-2,3-oxidosqualene to determine
the stereochemistry at C-17 of lanosterol from the reaction
catalyzed by 2,3-oxidosqualene-lanosterol cyclase
B:
H
extra double bond
OH
H
3H
oxidosqualene
cyclase
yeast
HO
O
13.44
Scheme 13.20
20
3H
13.45
20
3H
Me
Me
17
Me
H
HO
H
OH
Me
H
17
Me
H
17
H
13.46
Me
Model Study for Stereospecificity and
Importance of 17 Configuration
Chemical model for the conversion of
protosterol to lanosterol
Scheme 13.21
BF3
OH
H
Me
B:
H
H
Me
H
BzO
17
H
Me
Me
Me
13.47
H
BF3
H
CH2Cl2
-90°C
3 min
90%
H
Me
Me
BzO
H
13.48
With the 17 isomer a mixture
of C-20 epimers is formed
17
Evidence that the Cyclization Is Not Concerted
Mechanism proposed for the formation of the minor product isolated in the 2,3oxidosqualene cyclase-catalyzed reaction with 20-oxa-2,3-oxidosqualene
O
O
H
X
H
enzyme
+
H
H
X=O
HO
HO
13.43
40%
H+
Markovnikov addition
not when
X=CH2
X
H
X=O
a
b
13.49
3%
H
H
HO
HO
H
O +
a
H
H
H
13.41
O
H
does not
come from
a concerted
reaction
H
H 13.50
13.51
X = CH2 or O
ring expansion
b
X
X
+
+
X=O
H
HO
H
HO
H
13.52
13.43
H
13.42
Scheme 13.22
Evidence for Carbocation Intermediate
R
6
7
O
B
H
13.53
no reaction without methyls suggests initial epoxide opening
Vmax/Km for R = CH3, H, Cl
138, 9.4, 21.9 pmol g-1h-1M-1
correlates with carbocation stabilization (CH3 > Cl >H)
Squalene Biosynthesis
Squalene synthase-catalyzed conversion of
farnesyl diphosphate to squalene via presqualene
diphosphate
R
R
H
3-O P O
6 2
-H+
R
-PPi
R
3-O P O
6 2
NADPH
3-O P O
6 2
H
13.54
farnesyl diphosphate
NADP+, PPi
R
13.55
presqualene diphosphate
R=
Scheme 13.23
R
13.37
squalene
Rearrangement of Presqualene Diphosphate
to Squalene
Mechanism proposed for the conversion of
presqualene to squalene by squalene synthase
R
R
R
H
R
H
H
H2C
H
3-O P O
6 2
NADP H
13.55
R
R=
Scheme 13.24
R
13.56
R
13.57
NADP+
H
R
squalene
In the Absence of NADPH there is a Slow Hydrolysis
Evidence for 13.56 and 13.57
Mechanisms proposed for the squalene synthasecatalyzed hydrolysis of presqualene diphosphate to
several different products in the absence of NADPH
H
c,d
H
b
H
H2C
3-O P O
6 2
b
a
R
13.56 a
H
R
13.57
OH
d
c
B:
a
R=
OH
B:
R
13.55
d
c
H
R
R
R
R
R
R
R
R
H
R
HO
R
OH
Scheme 13.25
13.58
14%
R
13.59
58%
13.60
24%
Support for Intermediate 13.57
Use of dihydro-NADPH to provide evidence for the formation
of intermediate 13.57 in the reaction catalyzed by squalene
synthase
dihydro-NADPH
R
H H
H
H
B:
HO
N
R
R
13.57
R=
H
NH2
OH
R
O
13.61
unreactive NADPH
to mimic bound NADPH
R
13.62
Scheme 13.26
Rearrangements Via Radical Intermediates
DNA Photolyase
UV light causes DNA damage
Reactions catalyzed by DNA photolyase and (6-4) photolyase
O
O
O
DNA photolyase
HN
O
NH
N
N
O
HN
NH
h (visible)
O
P
13.63
cyclobutane pyrimidine dimer
h (uv)
O
N
visible h used as
a substrate for
photoreactivation
N
O
P
O
O
HN
O
OH
N
O
(6-4) photolyase
N
O
HN
h (visible)
NH
H
N
P
13.64
(6-4) photoproduct
h (uv)
both types
carcinogenic,
mutagenic
O
N
N
O
P
Scheme 13.27
NH2
Other Cofactors Used by Photolyases
O
CH2O
O
O-
P
N
O
OHO
N
N
O CH2
CH2
N
N
O
P
(CHOH)3
N
OH
O
NH
N
H
O
H2N
N
H
N
13.65
reduced FADH-
H
HN
N
O
O
N
13.66
N5,N10-methenyl H4PteGlun
C
CH2OH
O
H
N
CHCH2CH2 C
COO-
(CHOH)3
OH
n
CH2
HO
These act as photoantennae to absorb
blue light and transmit to the FADH-
N
N
O
NH
13.67
O
8-OH-7,8-didemethyl-5-deazariboflavin
Mechanism Proposed for DNA Photolyase
H2N
N
H
N
h (300-500 nm)
H2N
H
HN
O
O
HN
O
N
HN
N R
N R
13.66*
*
R'
O
N
P
13.63
O
N
O
13.66
NH
N
H
N
O
N
*
H
N
N
N
H
O
13.65*
O
R'
O
N
NH
N
H
N
O
NH
O
13.65
R'
HN
NH
N
O
N
N
O
N
H
P
O
N
NH
O
13.68
O
HN
O
O
O
HN
NH
N
N
O
O
O
O
HN
NH
N
N
O
O
O
NH
N
N
P
P
P
13.69
13.70
13.71
EPR evidence
O
Scheme 13.28
Proposed Mechanism for the Formation of
the (6-4) Photoproduct
O
O
O
H
HN
O
O
N
H
N
O
N
P
HN
h (uv)
[2+2]
O
HN
N
O
N
H
P
13.72
Scheme 13.29
H
OH
O
N
N
O
N
H
P
13.64
O
N
O
HN
O
O
N
H
:B
N
O
H
Mechanism Proposed for (6-4) Photolyase
H2N
N
H
N
N
h (300-500 nm)
H
HN
P
13.64
N R
O
HN
O
N
H
N
N
O
N
NH
N
H
O
N
O
NH
O
13.65
R'
HN
O
N
R'
13.65*
O
O
N
N
H
P
13.72
N R
*
R'
O
Scheme 13.30
N
O
13.66*
13.66
H
O
N
N
H
HN
N
O
H2N
*
H
N
H
N
H
N
O
N
O
N
H
N
NH
O
P
O
O
HN
O
O
N
H
N
HN
O
O
O
H
O
N
N
H
H
N
O
N
O
HN
O
NH
N
N
P
P
P
13.71
O
Coenzyme B12 Rearrangements
adenosylcobalamin
O
H2NC
CONH2
H2NC
A
O
N R
R
CoIII
H
D
H2NC
B
N
N
CONH2
OH OH
N
C
a
O
O
CH2
HN
N
O
CONH2
N
H
O
b
O
O
P
O
H
13.73
N
N
N
NH2
HO
-O
(coenzyme B12)
N
OH
H2O
(vitamin B12)
R
CH2
cobalamin ring
5-deoxyadenosyl
Co
13.74
abbreviation for coenzyme B12
Conversion of Vitamin B12 to Coenzyme B12
Bioynthesis of coenzyme B12
H2O
CoIII
NADH/FAD
NADH/FAD
CoII
cob(III)alamin
reductase
cob(II)alamin
reductase
B12r
O
O
=O P O P O P O
3
OCoI
13.75
Scheme 13.31
B12s
O-
CH2
O
Ad
2nd known
reaction at C-5
of ATP
-P3O10-5
OH
OH
adenosylating
enzyme
Mg2+
R
CH2
Co
Light Sensitivity of the Co-C Bond
of Coenzyme B12
R
CH2
Co
RCH2 is 5'-deoxyadenosyl
Scheme 13.32
h
CoII
13.76
R
+
CH2
13.77
Table 13.1. CoenzymeB12 -Dependent Enzyme-CatalyzedReactions
Enzyme
Reaction Catalyzed
CARBON SKELETAL
REARRANGEMENTS
CH 3
Methylmalonyl-CoA mutase
HOOC
HOOCCH 2CH 2 COSCoA
CH COSCoA
CH 3
2-Methyleneglutarate
mutase
HOOC
HOOCCH 2CH 2 C COOH
CH C COOH
CH 2
CH 2
CH 3
Glutamate mutase
HOOC
HOOCCH 2CH 2 CH COOH
CH CH COOH
NH2
NH2
Isobutyryl-CoA mutase
CH 3
H3C
CH 3CH 2CH 2 COSCoA
CH COSCoA
ELIMINATIONS
Diol dehydratase
R CH CH 2OH
OH
Glycerol dehydratase
RCH 2CHO
R = CH 3 or H
HOCH 2 CH CH 2OH
HOCH 2 CH 2CHO
OH
Ethanolamine ammonia lyase
CH 3CHO
CH 2 CH 2OH
NH2
ISOMERIZATIONS
L-b-Lysine-5 ,6-aminomutase
H2C CH 2 CH 2 CHCH 2 COOH
NH2
D-Ornithine-4 ,5-aminomutase
H3C CH CH 2 CHCH 2 COOH
NH2
NH2
H2C CH 2 CH 2 CH COOH
NH2
H3C CH
NH2
CH 2 CH COOH
NH2
NH2
NH2
REDUCTION
Ribonucleotide reductase
4-
O9 P3O
O
OH
N
4-O P O
9 3
reductant
OH
O
OH
N
General Form of Coenzyme B12-Dependent
Rearrangements
H
C1
C2
H
Y
X
C1
C2
Y
X
X is alkyl, acyl, or electronegative group
Scheme 13.33
Three Examples of Coenzyme B12
Rearrangements
Figure 13.2
A
HOOC
glutamate
mutase
H
CH2
C
CH
H
H
COOH
H CH2
C
HOOC
NH2
CH3 CH
C
OH
H
OH
diol dehydratase
CH2
C
NH2
H
OH
CH3 CH C
H
O
OH
-H2O
H
H
C
CH
NH2
H
B
COOH
CH2 CHCOONH3+
D-ornithine 4,5aminomutase
H CH2 CHCH2 CHCOONH2
NH3+
CH3CH2C
H
Mechanism for Diol Dehydratase and Ethanolamine
Ammonia-Lyase
Stereospecific conversion of (1R,2R)-[1-2H]-[1-14C]propanediol
to (2S)-[2-2H]-[1-14C]propionaldehyde catalyzed by diol
dehydratase
CH3
HO
HO
C
14
R
H
R
C
CH3
diol dehydratase
H
C
D
D
14
C
H
H
O
13.78
13.79
(1R, 2R)
(2S)
Scheme 13.34
No incorporation of solvent protons; therefore no elimination
of water (enol would form)
Stereospecific [1,2] migration of the pro-R H with inversion
kH/kD = 10-12
Stereospecific Conversion of (1R,2S)-[1-2H]-[1-14C]propanediol
to [1-2H]-[1-14C]propionaldehyde Catalyzed by Diol
Dehydratase
CH3
S
H
HO
C
14
R
C
CH3
OH
diol dehydratase
H
C
H
14
D
(1R, 2S)
C
D
O
H
13.80
13.81
Scheme 13.35
With the (1R, 2S) epimer, the pro-S H migrates; therefore
stereochemistry at C-2 determines which C-1 H migrates
Stereospecificity of Elimination of Water
Diol dehydratase-catalyzed conversion of (2S)-[1-18O]propanediol to
[18O]propionaldehyde (A) and of (2R)-[1-18O]propanediol to propionaldehyde (B)
CH3
CH3
H
C
OH
H18O
C
HR
A
HS
H
C
H
migrates
HO
C
H
pro-R
HS
H18O
CH3
- H2O
(pro-R hydroxyl
group loss)
H
C
C
18O
pro-S
13.82
H
H
13.83
(2S)-[1-18O]
CH3
HO
C
CH3
H
B
H18O
C
HR
HS
HR
migrates
H
H18O
pro-R
C
C
OH
-
H
CH3
H218O
(pro-R hydroxyl
group loss)
H
pro-S
H
C
H
C
O
H
13.84
(2R)-[1-18O]
Scheme 13.36
The same OH is eliminated (pro-R) regardless of which C-1 H migrates
Therefore the C-1 H and the C-2 OH migrate from opposite sides
giving inversion at both C-1 and C-2
Crossover Experiment to Show that Diol
Dehydratase Catalyzes an Intermolecular
Transfer of a Hydrogen from C-1 to C-2
CH3
H
HO
C
C
OH
H
H2C
+
OH
CH3
diol dehydratase
H
C
H
3H
+
H
C
3H
HO CH2
3H
13.85
C
O
C
H
O
Scheme 13.37
Therefore, hydrogen transfer is intermolecular
H
1
2
i
n
C
o
e
n
yz
m
e
B
R
a
d
io
a
ct
vi
it
y
Time Course for Incorporation of Tritium
from [1-3H]propanediol into the Cobalamin
of Diol Dehydratase
0
Figure 13.3
30
Time (sec)
60
Determination of the Site of Incorporation of 3H
into Coenzyme B12
Aerobic and anaerobic photolytic degradation of coenzyme B12 to locate
the position of the tritium incorporated from [1-3H]propanediol in a
reaction catalyzed by diol dehydratase
OH OH
aerobic
OH OH
h
1/2 3H lost
OH
O
+
Co
O
no 3H here
O
CH2
N
N
C
N
H
N
13.86
3H
here
Co
N
N
NH2
N
OH OH
NH2
OH
anaerobic
Scheme 13.38
N
all 3H retained
O
+
Co
h
H2C
no 3H here
3H
here
N
N
13.87
N
N
NH2
Reconstitution of the isolated [3H] coenzyme B12 into apoenzyme with propanediol
gives [2-3H]propionaldehyde. All 3H transferred from [3H] coenzyme B12
Synthesized (R,S)-[5-3H] Coenzyme B12
Transfers All 3H to the Product Randomly
OH
OH
O
CH3
N
N
13.88
N
N
NH2
possible intermediate to
equilibrate the C-5 protons
13.88 isolated with substrates that cannot rearrange
Coenzyme B12 is the hydrogen transfer agent.
Proposed Rationalization for EPR
Spectrum of Co(II) + Carbon Radicals
Formation of 5-deoxyadenosine, cob(II)alamin, and
substrate radicals during coenzyme B12-dependent
reactions
R
Substrate-H
+
H C H
CoIII
Scheme 13.39
R
H
C
H
+
H
CoII
Substrate
Product
Mechanism(s) Proposed for Diol Dehydratase
The part shown in the dashed box is even more speculative
than the rest of the mechanism
Not clear if
important
Scheme 13.40
OH CH3
R
R
H
CH2
CH2
Co
CH
CH
R
OH
OH CH3
CH3
13.88
CH
CH OH
13.89
OH CH3
R
CH3
Co
Co
CH
CH OH
Co
13.92
O
H C CH2CH3
H2O
OH CH3
R
HO CH CH2
13.91
CH2
Co
OH CH3
R
HO
CH
CH
13.90
CH2
OH CH3
R
HO CH
CH
CH3
Co
H
Co
Radicals observed in EPR spectrum
13.93
Chemical Model Study for a Proposed Diol
Dehydratase-catalyzed Rearrangement
Involving a Co(III)-olefin -Complex
The trapezoid represents the cobaloxime ligand
CH2
13CH
2
Co
N
CH2
Co
13CH
2
OAc
MeOH
N
13CH
CH2
Co
N
2
13.96
MeO CH2
13CH
2
Co
13.94
Scheme 13.41
13.95
N
13.97
OMe
A Cobalt Complex Is Not Necessary
The Fenton reaction as a model for a
proposed diol dehydratase-catalyzed
free radical rearrangement
HO CH2
CH2
Fe2+ + H2O2
HO CH
CH2
X
Fe3+ +
X
+
+ H2O2 + Fe2+
CH3CHO
HO
HO CH
HO
CH2
X
H+
H
CH2
XH
-XH
Fe+3
O CH
HO CH
CH3
Scheme 13.42
Fe+2
HO CH
CH2
HO CH
CH2
HO CH
CH2
Another Chemical Model Study for a Proposed Diol
Dehydratase-catalyzed Free Radical Rearrangement
(the cobalt complex is just to initiate the
reaction by radical generation)
OH
OH
OH
OH
h
Co
OH
OH
or 
N
13.100
H
13.99
OH
13.98
OH
O
13.101
OH
-H2O
H
Scheme 13.43
OH
H
OH
OH
Carbon Skeletal Rearrangements
Stepwise (a) versus concerted (b) mechanisms for the methylmalonyl-CoA
mutase-catalyzed generation of 5-deoxyadenosine, cob(II)alamin, and
substrate radical
Scheme 13.44
H
H
O
O
SCoA
R H
CH2
H
COO-
R
O
H
R
SCoA
H
CH2
H
Co
Co
Co
N
N
N
N
H
610His
DMB
610His
N
H
DMB
H
H
CH3
COO-
b
a
DMB
H
N
H
SCoA
H
COO-
610His
*
b
EPR confirms Co(II) + organic radical
Crystal structures with and without substrates bound show the active site closes
upon substrate binding - shields radical intermediates
Co-C cleavage is 21 times faster with (CH3)MM-CoA than with
(CD3)MM-CoA. Therefore, Co-C and C-H cleavage are concerted.
O
Six Possible Pathways for the
Conversion of MethylmalonylCoA Radical to Succinyl-CoA
Radical Catalyzed by
Co(I)
Methylmalonyl-CoA Mutase Co(II)
H
H
SCoA
H
H
COO13.104
COO-
H 13.105
O
SCoA
O
H
H
a
SCoA
O
H
SCoA
H
H
COO-
COO-
H 13.107
13.106
Co(III)
Co(II)
Co(I)
H
Co(III)
Co(II)
b
O
H
H
Co(II)
SCoA
O
H
c
SCoA
H
H
COO13.102
d
O
H
e
H
H
SCoA
COO13.103
H
COO13.108
O
f
O
Co(II)
13.110
SCoA
H
H
H
COO-
SCoA
Co(II)
H
H
H
COO-
13.109
O
Ab initio calculations disfavor
pathway e
No concensus about the others
O
H
H
SCoA
H
COOCo
13.111
H
SCoA
H
COO-
H
Co
13,112
Figure 13.4
Ribonucleotide Reductase
Converts ribonucleotides to deoxyribonucleotides
Results are different from other coenzyme B12 enzymes:
• 0.01-0.1% of 3H from [3-3H]UTP is released
• no 3H from [3-3H]UTP found in adenosylcobalamin
• no crossover between [3-3H]UTP + ATP
• [3-3H]UTP gives [3-3H]dUTP
• 3H in [5-3H]adenosylcobalamin is washed out in the
absence of substrate
• adenosylcobalamin  5-deoxyadenosine + Co(II)
By EPR formation of Co(II) corresponds to formation
of 5-deoxyadenosine and the generation of a thiiyl radical
(Cys-408)
Mechanism Proposed for
Coenzyme B12-dependent
Ribonucleotide Reductase
C408
S
S
H
CH3Ado
CH2Ado
•
CoII
rates of formation
are identical;
therefore,
concerted reaction
Co
Scheme 13.45
His
4-O P O
9 3
Ha
S
S
S
B
O Hb
Ha
O
4-O P O
9 3
Hb
•
OH
B-
O
OH
SH
SH
H
B-
OH
H
S
SH
-H2O
O
BH S
S
S
4-O P O
9 3
Ha
O
Hb B
Hb
SH
S
Ha
O Hb B
4-O P O
9 3
B
13.114
13.113
C119 C419
Ha
O
4-O P O
9 3
B
Ha
4-O P O
9 3
O Hb
•
HO
B-
H
S
13.117
S
B-
HO
H
S
S
13.116
H
B
O
H
S
13.115
S
B
Mechanism Proposed for Reducing and
Reestablishing the Active Site of Coenzyme
B12-dependent Ribonucleotide Reductase
C408
C408
S
Scheme 13.46
S
H
H
CH2Ado
CH2Ado
•
CoII
Co
His
B-
electrons are
transferred to
active-site
disulfide
S
S
C736
SH
SH
C119 C419
13.117
SH
B-
SH
C731
S
C736
regenerates active
site for next cycle
S
C731
reduced by
thioredoxin
The function of the cobalamin in this enzyme is to initiate
the radical reaction by abstraction of H• from Cys-408
Other Ribonucleotide Reductases Use Other Radicals
to Abstract a H• from an Active Site Cys
Cofactors for class I (13.118), class III (13.119), and class IV
(13.120) ribonucleotide reductases
O
122Tyr
O
O
84Asp
O
H2O
H2O
O
Fe
O
H3N
238Glu
O
O
O
115Glu
241His
S
Fe
O
Fe
S
Fe
S
204Glu
H3C
S
Fe
O
Mn
13.120
Ade
OH OH
13.119
Tyr
O
S
13.118
Figure 13.5
H
NH
Fe
O
118His
CO2
O
Mn
Reaction Catalyzed by
Lysine 2,3-Aminomutase
pro-R
H
H3N
NH3+
COO-
Ha Hb
13.121
L--Lys
SAM
[4Fe-4S]+2
PLP
Hb
COO-
H3N
H3N
pro-R
H
Ha
13.122
L--Lys
Scheme 13.47
Requires PLP, SAM, [4Fe-4S], and a reducing agent
Transfers 3-pro-R H of L--Lys to 2-pro-R of L--Lys with
migration of 2-amino of L--Lys to C-3 of L--Lys
No exchange with solvent
With (S)-[5-3H]adenosylmethionine, 3H ends up in both L-Lys and L--Lys
One equivalent of Met and 5-deoxyadenosine are formed
with L--[3-3H]Lys. 1-6% of 3H ends up in SAM
C-S bond is stable, unlike C-Co bond
In the presence of a reducing agent, [4Fe-4S]+ is observed
in the EPR, which reduces SAM to Met and
5-deoxyadenosyl radical
It appears that SAM is functioning like coenzyme B12
Mechanism Proposed for Lysine 2,3-Aminomutase
O
COOH +
H3N
=O
OH
3PO
[4Fe-4S]2+
e-
=O
H
S
NH3+
3PO
N
H
13.123
Ado
Ado CH2
H
H
H3N
H
N
H
H3 N
=O
N
H
13.126
H
Scheme 13.48
H
COO-
NH2
13.122
+ PLP + SAM +
-OOC
[4Fe-4S]+
CH3
H
N
H
N
H
CH3
OH
3PO
OH
3PO
CH3
Met
[4Fe-4S]2+
H2 O
H3N
CH3
=O
=O
13.127
H
N
OH
3PO
N
H
13.124
H3 N
COOH
CH3
Ado
H
COO-
OH
3PO
not observed
in EPR
CH2
Met
[4Fe-4S]2+
CH2
=O
OH
COO-
[4Fe-4S]+
Ado
N
HR HS
CH3
CH2
COOH
CH3
H3 N
N
N
H
Ado
Ado
H
H3N
NH2
13.121
COO-
13.125
N
H
CH3
unique function
for PLP
EPR detects organic radicals;
13C label shows product radical
13.126 in EPR spectrum
Model Study for New Function of PLP
Chemical model study to test the proposed
rearrangement mechanism for lysine 2,3aminomutase
CH3
CH3
Br
Bu3Sn
CO2Et
N
Bu3SnH
AIBN

CO2Et
N
Ph
Ph
- Bu3Sn
CH3
CH3
CO2Et
N
Ph
Scheme 13.49
CH3
CO2Et
CO2Et
N
N
Ph
H
Ph
SnBu3
To Get Evidence for Substrate Radical (13.124)
stabilize -radical
COOS
H3N
13.128
H
NH2
Evidence for Substrate Radical Formation
Lysine 2,3-aminomutase-catalyzed rearrangement of
4-thialysine to generate a more stable substrate radical
COOH
S
H3N
H
S
H3 N
N
HR HS
S
H3N
N
H3N
H
13.129
Pyr
Pyr
Pyr
13.130
+
S– NH3
EPR detected
Ado–CH2
Ado–CH2
COO-
PLP
+
O
+
H
13.131
COO-
HO
NH4+
H2 O
N
Ado–CH3
Ado–CH3
Pyr
Pyr = pyridine ring of PLP
COO-
S
N
Ado–CH3
Ado–CH2
COO-
COO-
COO-
H
HO
N
COOH
OH
+
N
N
Pyr
B:
Pyr
Pyr
isolated
Scheme 13.50