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The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 5
Dioxygenation
Dioxygenases
Incorporation of both atoms of O2 into substrates
Intramolecular dioxygenases (both
atoms of O2 into same molecule)
Different types of aromatic cleavages
for catechol dioxygenases
contain nonheme FeIII (EPR) red
C
OH
A
R
OH
B
A — intradiol cleavage
B — proximal extradiol cleavage contain nonheme
C — distal extradiol cleavage
II
Fe (no EPR)
colorless
Figure 5.1
Intermolecular dioxygenases (two O atoms of O2 go into two
different products)
A Mechanism for Intradiol Dioxygenases Using
Protocatechuate 3,4-Dioxygenase as an Example
COO
Tyr447
ON
Fe3+ O
Tyr447
O
His460 N
Fe3+ O
H
Tyr408 O
462
N His
Arg457
O
Tyr447
H
+ substrate
N
- substrate
H
O
N
5.1
OH O
O
O-
N
OH
Fe3+
COO
Tyr447
O
Fe3+
O
H
O
H
O
N
5.2
N
H
NH
N
H
COO
Arg457
NH2
COO
NH
N
H
O
NH2
O
Fe2+
Fe2+
O
O
N
O
N
COO
O
NH2
H
O
O
Fe3+
O
O
O
N
O
N
O
Tyr447
COO
NH
O
N
O O
N
Arg457
N
H
O
Fe3+
+O2
O H
O
O
N
N
H
5.3
5.4
5.5
Tyr447
COO
O
COO
O
O
O
O
N
5.6
O
Fe3+
O
O
N
Fe3+
O
O
N
N
5.7
5.8
Criegee rearrangement
[1,2] shift to peroxide
His460
O
O
O
N
Fe3+
COO
O
O
O
N
H
H
O
H
Scheme 5.1
HOOC
HOOC
5.9
COO
Tyr447
O
N
Fe3+ O
Tyr408 O
H
N His462
Mechanism is based on model
studies and crystal structures
Alternate Mechanism
Criegee versus dioxetane mechanisms for
intradiol dioxygenases
Catechol 1,2-dioxygenase
Fe
H
HO
O
Criegee**
OH
Fe
CO
H
CO
2
OH
a
O
HO
OH
5.11
5.12
Product found
to have lost
some 18O (not
from washout)
OH
OH
5.10
2
O
b
CO
CO
dioxetane
Scheme 5.2
O
5.13
OH
This would
have retained
both 18O atoms
A Mechanism for Extradiol Dioxygenases Using 2,3Dihydroxyphenylpropionate 1,2-Dioxygenase as an Example
CO2-
CO2-
CO2O
O
O
OH
OH
O2
FeII
O
O
b
O
O
O
FeII
O
O
5.15b
5.15a
5.15c
a
b
B
CO2-
CO2-
O
O
O
O
FeII
FeII
O
O
5.18
CO2-
5.16
B
Criegee CO
2
H
O
O
O
O
O-
Scheme 5.3
OH
OH
FeII
O
5.20
nucleophilic
addition
 to C-O
O
H
O
O
O
O
FeII
O
CO2-
O
a
O
FeII
FeII
O
5.14
CO2-
CO2-
5.19
O
O
FeII
O
5.17
Evidence for Criegee as in Scheme 5.2
Evidence for a Radical Intermediate in Extradiol
Catechol Dioxygenases
CO2-
Scheme 5.4
CO2-
H
H
CO2-
H
H
OH
OH
O
FeII
OH
OH
O
5.21
5.22
“Reversible”
O2
O2
CO2-
CO2-
H
CO2-
H
H
H
O
O
O
FeII
O
O2C
OH
5.23
5.25
O2C
OH
5.24
Both diastereomers give the same ratio of 5.23 and 5.24
Consistent with a reversible cyclopropylcarbinyl radical opening
Reactions Catalyzed by Prostaglandin H
Synthase (Cyclooxygenase)
Heme-dependent enzyme
with or without the double bond
5
with or without the double bond
18O
18O
COO+ 2
18O
2
5.26
Scheme 5.5
COO-
COO18O
18O
arachidonic acid
(with double bond)
with or without the double bond
H
H
18O18OH
5.28
PGH
5.27
PGG
cyclooxygenase
activity
18OH
peroxidase
activity
Conversion of Prostaglandin H to Other
Prostaglandins
with or without the double bond
18O
18OH
with or without the double bond
18O
with or without the double bond
9
COO-
COO-
COO-
+
15
18O
18O
H
H
18OH
5.28
PGH
H18O
18OH
H
5.29
PGD
18OH
18OH
5.30
PGE
with or without the double bond
COO-
COO18O
H18O
H
18OH
5.31
PGFa
Scheme 5.6
H18O
H
5.32
PGI
18OH
Prostaglandin H Synthase-catalyzed Incorporation
of 18O2 into 8,11,14-Eicostrienoic Acid
18O
9
8
9
COO+ 2 18O2
-H218O
11
13
11
12
COO-
8
14
5.33
Scheme 5.7
H18O
12
13
H
18OH
PGE1
3 18O atoms
Mixture of 18O2 + 16O2 gives both ring O’s as 18O or 16O;
therefore from same molecule of O2
H’s at C-8, C-11, C-12 retained
H at C-9 lost in PGE, and retained in PGF1
Only pro-S H at C-13 removed
A Hydroxylated By-product Isolated from the
Prostaglandin H Synthase-catalyzed Reaction
COO-
COO-
HO
•OO
Scheme 5.8
5.34
isolated as a byproduct
Therefore, endoperoxidation occurs
prior to hydroperoxidation at C-15
Further Evidence that
Endoperoxidation Occurs First
COO-
HOO
H
5.35
No PGE formed from this
Two Possible Mechanisms for Prostaglandin H
Tyr-385
X3H
X
3
a H
H
COOH
a
O
pro-S (3H•)
COOH
H
O
COOH
H
O
O
5.36
H
O
5.37
O
H
O
3H
H
O
COOH
COOH
O
pro-S (3H•)
b
O
H
Scheme 5.9
Tyr-385
O
3H
X
5.38
X
COOH
O
O
O
H
H
X
OOH
PGG1
X
Evidence for pathway a: intermediate found containing one O2 and no 3H
Tyr385Phe has peroxidase activity, but no cyclooxygenase activity
Peroxidase active site is adjacent to cyclooxygenase active site - peroxidase
regenerates protein radical in cyclooxygenase ( X•) and it reduces PGG to PGH contains ferric heme
Iron-oxo species from heme initially abstracts H• from Tyr-385 ( X-H)
Conversion of Prostaglandin H to
Prostaglandin E and F
:B
H B
H
COOH
9
O
no C-9 H
O
COOH
isomerase
(A)
O
H
B+
H
HO
OH
H
OH
:B
PGH1
PGE1
C-9 H retained
:B
:B
H
GS H
H
GS H
COOH
O
GS
O
B+
H
COOH
HO
H
COOH
reductase
(B)
O
H
HO
B+
OH
H
PGH1
Scheme 5.10
H
OH
HO
H
OH
PGF1a
Intermolecular Dioxygenases
Reaction catalyzed by -keto acid-dependent
dioxygenases
like heme enzymes
O
Substrate-H
+
18O
2
O
+ R
E-Fe2+
ascorbate
O
usually -ketoglutarate
Substrate-18OH
+
R
18O
+ CO2
(or cyclized product
+ H218O)
keeps iron reduced
Scheme 5.11
O
nonheme Fe2+
if -ketoglutarate,
then this is
succinic acid
Mechanism to Account for Formation of Succinic
Acid, CO2, and an Iron-oxo Species
Conversion of FeII and O2 to a high energy iron-oxo species
catalyzed by -keto acid-dependent dioxygenases
X
X
X
O
O
X
Fe2+
X
O
COO-
O
X
Fe3+
X
O
O2
X
O
O
Fe4+
O
O
O
COO-
O
O
Fe4+
X
O
COO-
O
X
X
O
O
X
O
O
COO-
from CD spectrum
COO-
-O
Scheme 5.12
O
X
X
+
CO2
+
X
Fe4+
X
O
5.39
like heme-oxo species
Early Mechanisms for Hydroxylation of Prolyl
Residues by Prolyl Hydroxylase (proposed in 1979)
O
L
2L + Fe2+ + O2
FeO2
+
COO-
-OOC
O-
O
O
-ketoglutarate
L
L = ascorbate
OH
-OOC
O
H
R1
N
H
L
+ O
R
O
L
H
O
HO
HO
Fe
L
L
O
O
R
H
Fe
L
O
R1
R1
N
H
N
H
R
O
Scheme 5.13
Fe
L
L
R1
N
O
Fe
O
O
-L
Fe
+
-OOC
H
H
L
COO-
O
H
H
-CO2
R
+
L + Fe2+
Mechanism for Clavaminate SynthaseCatalyzed Ring Closure
from -ketoglutarate
Fe2+ and O2
O
L
HO
FeIV
L
H
L IV
Fe O
L
OH
N
NH2
O
.
O
N
NH2
O
FeII
a
L
H
O
L
H
+
N
N
+
NH2
COO-
Scheme 5.14
NH2
COO-
O
O
L
N
b
HO
a O
L
O
COO-
COO-
FeIII
HO FeII
NH2
O
L
+H+
COO-
clavulanic acid
L
L
H2O
+
FeII
L
Reactions Catalyzed by Thymine Hydroxylase
Evidence for iron-oxo mechanism
D isotope effect w/CD3
O
CH3 -KG + O
2
HN
O
O
N
H
succinate + CO2
CH2OH -KG + O
2
HN
O
N
H
O
O
succinate + CO2 O
CHO
HN
N
H
-KG + O2
succinate + CO2
COOH
HN
O
N
H
5.40
Scheme 5.15
Reactions resemble heme-dependent enzymes
Other Alternative Substrates for Thymine
Hydroxylase
gets epoxidized
S-oxygenation
O
O
HN
O
5.41
O
O
SCH3
HN
N
H
hydroxylation
N
H
5.42
CH3
HN
O
O
N
H
5.43
CH3
HN
O
N
CH3
5.44
All similar to heme-dependent
P450 reactions
With 18O2
1 18O in succinate
1 18O in product
N-dealkylation
Inactivation of Thymine Hydroxylase by
5-Ethynyluracil
inactivates thymine hydroxylase, like P450
O
O
Fe(IV)=O Fe(II)
HN
O
N
H
5.45
O
O
O
HN
HN
O
O
a
O
N
H
N
H
HN
a
H
O
5.47
5.46
H2O
N
H
O
O
H
N
HN
carbene
insertion
HN
Gly
5.48
b
CH2
O
C O
O
N
H
CO2H
N
H
5.49
COOH
O
5.50
O
O
H
O
Fe(II)
OH
O
NH
CH2
Scheme 5.16
Fe(IV)=O
5.51
N
H
O
NH
CH2
5.52
N
H
O
from NMR after
tryptic digestion
Intramolecular Version of -Keto Acid
Dioxygenase
Reaction catalyzed by
(p-hydroxyphenyl)pyruvate dioxygenase
0.3 18O atom
partial exchange
with solvent
18O
2
HO
COO5.53
O
Scheme 5.17
Fe2+
18OH
HO
5.54
+ CO2
CO18O-
1 18O atom
Model Study for the Mechanism of
(p-Hydroxyphenyl)pyruvate Dioxygenase
OH
HO
+
O
O
C
1O
2
O
O
O-
O
COO-
5.55
+H+
COO-
HO
OH
CO2
COO-
pH 12
O
OH
5.56
Scheme 5.18
Evidence Against this Mechanism
• If 5.56 is incubated with the enzyme,
O
no product is formed
COOOH
5.56
•
R
COOH
5.57
O
R = H or F are substrates
• Enzyme also catalyzes sulfoxidations
like P450
Proposed Mechanism for
(p-Hydroxyphenyl)pyruvate Dioxygenase
Like heme peroxide
O2
X
O
FeII
X
O
O
O
OH
ascorbate
X
X
FeIII
X
O
O
FeIII
O
X
O
OH
O
O
OH
O-
O
O
-CO2
X
X
FeII
X
X
O
O
OH
O
electron
transfer
X
FeIII O
O
X
OH
O
O
OH
HO
5.58
X
OH
X
-O
O
OH
O
X
X
IV
Fe O
O
NIH
shift
FeII
exchange
with solvent
FeII
H
B
O
O
Scheme 5.19
Mechanism more like heme-dependent enzymes