chp13.ppt
TRANSCRIPT
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The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 13
Rearrangements
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Rearrangements
Pericyclic Reactions - concerted reactions in which bonding changes occur via reorganization of electrons within a loop of interacting orbitals
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Scheme 13.1
[3,3] sigmatropic rearrangement
General form of the Claisen rearrangement
O O
H
Sigmatropic Rearrangements
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Scheme 13.2
chorismate prephenate
Chorismate Mutase-catalyzed Conversion of Chorismate to Prephenate
COO-
HO
O COO-
CH2
HO
-OOC CH2 C
O
COO-
13.213.1
12
3
45
6
78
9 12
34
5
6
7
8
9
A step in the biosynthesis of Tyr and Phe in bacteria, fungi, plants
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Required conformer for Claisen rearrangement (10-40% observed in solution from NMR spectrum)
Conformation of Chorismate in Solution
O
-O2C
OH
COO-
13.6
chair-like TS‡
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Evidence for Chairlike Transition State
Scheme 13.3
Stereochemical outcome if chorismate mutase proceeds via chair and boat transition states, respectively, during reaction with (Z)-[9-3H]chorismate
O COO-
3H H
OH
COO-
O
-OOC
H3H
OH
COO-
OH
O
COO-
COO-H
3H
OH
3HH
COO-O
-OOC
Z-13.7
pro-S
chair
pro-R
boat
Z-13.7
13.8
13.9
A
B
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To Determine the Position of the 3H
Scheme 13.4
Z-[9- 3H]chorismate 20% 3H releaseE-[9- 3H]chorismate 67% 3H release
Therefore, chair TS‡
Chemoenzymatic degradation of the prephenate formed from the chorismate mutase-catalyzed conversion of (Z)-[9-
3H]chorismate to determine the position of the tritium
OH
-OOCHR
COO-
O
HS
HRO
COO-
HS HSOH
COO-
pH < 6
- CO2 , -H2O
phenylpyruvatetautomerase
-HR+
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Figure 13.1
2° inverse isotope effect on C-4 (sp2 sp3); therefore not 1-3 (sp3 sp2)
Five Hypothetical Stepwise Mechanisms for the Reaction Catalyzed by Chorismate Mutase
COO-
OH
O COO-
B+ H
COO-
O COO-
-OOC COO-
O
O
-OOCCOO-
O
-OOC
COO-
OH
COO-O
COO-
B+ H
OH
COO-O
COO-
HB:
O
COO-O
COO-
B+ H
B:
H X H:B
OH
COO-
-O
COO-
XB+H
O
COO-O
COO-
HO
COO--O
COO-
prephenate
prephenate
+
+
+
+
+
(1)
prephenate
prephenate
(2)
(3)
(4)
(5)
rearrangement
H2O
4
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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
Both are substratesCOO-
OCH3
O COO-
13.10
COO-
O COO-
13.11
Function of the enzyme is to stabilize the chair transition state geometry
Conclusion: pericyclic
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Oxy-Cope Rearrangement
Scheme 13.5
Cope
oxy-Cope
Neither observed yet by an enzyme, but a catalytic antibody has been raised
General form of Cope (A) and oxy-Cope (B) reactions
OH OOH
A
B
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Scheme 13.6
Oxy-Cope Rearrangement Catalyzed by an Antibody
COOH
HOOH
COOH
O
COOH
OH
COOH
OH
COOH
O
COOH
‡
13.13 13.14
13.15
bondrotation
bondrotation
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hapten to raise the antibody
OH
O
NH
ONH2
13.12
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Scheme 13.7
[2,3] Sigmatropic Rearrangement Catalyzed by Cyclohexanone Oxygenase
Ph Se PhSe
O O H
•
•O H
•
PhSeH
Oenzyme
NADPH
[2,3] sigmatropicrearrangement
13.16
13.17
PhSe
PhSe•
O2
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Scheme 13.9
boat like TS‡
[4+2] Cycloaddition (Diels-Alder) Reaction
R'
R"
R
R'
R"
RH
H
H
R
H
R"H
HR'
13.18
‡
(d,l)
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Scheme 13.10 solanopyrones
enzymaticexo : endo is 53 : 47
in aqueous solution exo : endo is 3 : 97 (nonenzymatic)
An Intramolecular Diels-Alder Reaction Catalyzed by Alternaria solani
O
OCH3
OHC
O O
OCH3OHC
O
O
OCH3
OHC
O H
H
H3C
O
OCH3OHC
O
H
H
H3C
exo endo
13.19a 13.19b
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Scheme 13.11
An Antibody-Catalyzed Diels-Alder Reaction
NH O COO-
O
N
O
O
NHAc
NH O COO-
O
N
O
O
NHAc
H
H+ NNH
O
O
NHAcO
O
-OOCH
H
‡
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Hapten used
N
NHO
O
NHAc
O
O
-OOCH
H
13.20
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This hapten gives an antibody that makes only endo product
This hapten gives an antibody that makes
only exo product
HN
O
O
O N
O
CONMe2
O
13.24
HN
O
O
O N
O
CONMe2O
13.23
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Rearrangements via a Carbenium Ion
Scheme 13.14
acid-catalyzed
acyloins[1,2] alkyl migration
An acid-catalyzed acyloin-type rearrangement
R C
O
C
OH
R"
R' R C
OH
C
OH
R"
R' R C
OH
C
O
R'
R"
H2O H
R C
OH
C
O
R'
R"
: ++
13.31
H: :
OH2
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Scheme 13.15
Reactions Catalyzed by Acetohydroxy Acid Isomeroreductase
CH3 C
O
C
OH
14CH3
COO- CH3 C
HO
C
OH
3H
COO-
14CH3
C
O
C
OH
14CH2CH3
COO- CH3 C
14CH2CH3
OH
C3H
OH
COO-
+ NADP++ NADP3H
+ NADP++ NADP3HCH3
13.32 13.33
13.34 13.35
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substrate
CH3 C
OH
C
O
COO-
CH3
13.36
Kinetically-competent intermediate
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Scheme 13.16 Proposed Acyloin-type Mechanism for Acetohydroxy Acid Isomeroreductase
CH3 C
O
C
O
R
COO-
H
CH3 C
OH
C
O
COO-
R
CH3 C
OH
C
OH
H
COO-
R
CH3 C
O
C
OH
R
COO-
B H
CH3 C
OH
C
OH
R
COO- CH3 C
OH
C
OH
R
COO-
CH3 C
OH
C
OH
R
COO-CH3 C
HO
C
O
R
COO-
H
CH3 C
OH
C
O
COO-
R
CH3 C
OH
C
OH
H
COO-
R
NADPH + H+
stepwise
++
+
+
:BB H
+
NADP+
::
NADPH + H+
NADP+
B:
concerted
intermediate
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CyclizationsSterol biosynthesis
Scheme 13.17
cholesterol
squalenelanosterol
Conversion of squalene to lanosterol
H18O
H
H
13.3813.37
10
10
NADPH
1
2
34
56
7
8
9
11
12
13
14
15
1617
20
18O2
O
squalene 2,3-epoxidase
NADPHO2, flavin,nonheme Fe2+
2,3-oxidosqualene-lanosterol cyclase
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17
HO
Me
Me
O
B+ H
XHO
H
HMe
H
X
Me MeB:
MeMe
H
MeMe13.39
:
3
21
9
1413
Me
20
H
Me
Me
H
8
13.40
13.38
MeMe
Me
H
Me
Scheme 13.18
2,3-oxidosqualene-lanosterol cyclasenot
isolated
17
protosterol
7 stereogenic centers
squalene 2,3-epoxidase
squalene
anti-Markovnikov (to get 6-membered ring)
Isotope labeling shows the 4 migrations are intramolecularCovalent catalysis proposed to control stereochemistry
Initial Mechanism Proposed for 2,3-Oxidosqualene-lanosterol Cyclase
(128 possible isomers) only isomer
formed
lanosterol
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Evidence for 17 Configuration
Scheme 13.19 no covalent catalysis needed
17
isolated
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
O
O
B+ H
HO
H
HMe
H
MeMe
HMe
Me
OMe
HO
H
HMe
H
13.41
13.43
MeMe
HMe
Me
O
13.42
Me
17
17
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Further Support for Structure of Protosterol
Scheme 13.20
17
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
HO
H
Me
3H
H
H
Me
O
3H
HO
Me
H
Me
3H
OH
H
H
Me
Me
17oxidosqualenecyclaseyeast
extra double bond
13.44
20
13.45
H OH
17
20
B:
13.46
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Model Study for Stereospecificity and Importance of 17 Configuration
Scheme 13.21
1717
90%
With the 17 isomer a mixture of C-20 epimers is formed
Chemical model for the conversion of protosterol to lanosterol
BzO
Me
H
Me
H
OH
H
H
Me
Me
BzO
Me
Me
H
H
H
H
Me
H
CH2Cl2-90°C3 min
13.47 13.48
BF3
B:
BF3
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O
X
O
HH
H
HHO
X
HO
H
HHO
H
O
H
HO
H
H
X
H
H
HOH
O
H
X = O
a
H
H
ab
b
HO
H
H
X
13.4113.49
40%
3%13.43
X = O
13.50
+
X = CH2 or O
13.52
13.51
+
+
+
X = O13.43
13.42
enzyme
H+
Evidence that the Cyclization Is Not Concerted
Scheme 13.22
Markovnikov additionnot when
X=CH2
ring expansion
Mechanism proposed for the formation of the minor product isolated in the 2,3-oxidosqualene cyclase-catalyzed reaction with 20-oxa-2,3-oxidosqualene
does not come from a concerted reaction
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Vmax/Km for R = CH3, H, Cl 138, 9.4, 21.9 pmol g-1h-1M-1
correlates with carbocation stabilization (CH3 > Cl >H)
Evidence for Carbocation Intermediate
O
R
B H13.53
6
7
no reaction without methyls - suggests initial epoxide opening
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R
3-O6P2O R
H
H
R
3-O6P2O
R
-PPi
R
R
NADPH
NADP+, PPi
13.3713.54
13.55
R =
3-O6P2O -H+
Squalene Biosynthesis
farnesyl diphosphatepresqualene diphosphate squalene
Squalene synthase-catalyzed conversion of farnesyl diphosphate to squalene via presqualene diphosphate
Scheme 13.23
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Rearrangement of Presqualene Diphosphate to Squalene
Scheme 13.24
squalene
Mechanism proposed for the conversion of presqualene to squalene by squalene synthase
13.55
R
H
3-O6P2O
RR
H
H2C
R RH
R
R
H
H
R
NADP H
R =
NADP+
13.56 13.57
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R
H
3-O6P2O
RR
H
H2C
R RH
R
H OHB:
OH
R
R
13.60
H OH
58%14%
B:
13.55
R
R
HO
HR =
13.56 13.57
13.58 13.59
R
R
c
24%
R
R
a
c,d d
a
a
b b
c
d
In the Absence of NADPH there is a Slow HydrolysisEvidence for 13.56 and 13.57
Scheme 13.25
Mechanisms proposed for the squalene synthase-catalyzed hydrolysis of presqualene diphosphate to several different products in the absence of NADPH
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Support for Intermediate 13.57
Scheme 13.26
dihydro-NADPH
Use of dihydro-NADPH to provide evidence for the formation of intermediate 13.57 in the reaction catalyzed by squalene synthase
RH
R13.62
H OH
R =
13.61
B:
13.57
R
H
R
HO
N
NH2
O
R
HH
unreactive NADPHto mimic bound NADPH
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HN
N N
NH
O O
O O
DNA photolyase
h (uv)
h (visible)
P
HN
N N
NH
O O
O O
P
13.63
HN
NO
O
P
OH
H
N
N O HN
N N
NH
O O
O O
P
(6-4) photolyase
h (uv)
h (visible)
13.64
DNA Photolyase UV light causes DNA damage
Reactions catalyzed by DNA photolyase and (6-4) photolyase
Scheme 13.27
visible h used as a substrate for photoreactivationcyclobutane pyrimidine dimer
(6-4) photoproduct
both types carcinogenic, mutagenic
Rearrangements Via Radical Intermediates
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reduced FADH-
N5,N10-methenyl H4PteGlun
8-OH-7,8-didemethyl-5-deazariboflavin
These act as photoantennae to absorb blue light and transmit to the FADH-
Other Cofactors Used by Photolyases
CH2
(CHOH)3
CH2O P
O
O
O-
P
O
O-
O CH2O
HO OH
N
N
N
N
NH2
NH
N
NH
N O
O
13.65
CH2
(CHOH)3
CH2OH
N
NH
NHO O
O13.67
HN
N
N
HN
N C
O
CHCH2CH2
COO-
H2N
O
H
C
O
OHHN
n13.66
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Scheme 13.28EPR evidence
Mechanism Proposed for DNA Photolyase
HN
N N
NH
O O
O O
P
HN
N N
NH
O O
O O
P
HN
N
N
HN
RN
H2N
O
H
HN
N
N
HN
RN
H2N
O
H
NH
N
NH
N O
O
R'
NH
N
NH
N O
O
R'
HN
N N
NH
O O
O O
PNH
N
NH
N O
O
R'
HN
N N
NH
O O
O O
P
HN
N N
NH
O O
O O
13.66
*h (300-500 nm)
13.65
*
P
13.63
13.68
13.69 13.70 13.71
13.66*
13.65*
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Scheme 13.29
Proposed Mechanism for the Formation of the (6-4) Photoproduct
HN
NO
O
P
OH
H N
N O
13.72
h (uv)
13.64
HN
NO
O
P
O
H N
N O
HHN
NO
O
P
O
H N
N O
H
[2+2]
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Scheme 13.30
Mechanism Proposed for (6-4) Photolyase
HN
N N
NH
O O
O O
P
HN
N
N
HN
RN
H2N
O
H
HN
N
N
HN
RN
H2N
O
H
NH
N
NH
N O
O
R'
NH
N
NH
N O
O
R'
NH
N
NH
N O
O
R'
HN
NO
O
P
O
H N
N O
HN
NO
O
P
O
H N
N O
H
H :B
HN
NO
O
P
O
H N
N O
H
HN
NO
O
P
O
H N
N O
H
*h (300-500 nm)
13.65
HN
NO
O
13.72
13.64
P
13.66
O
H N
N O
H
*
13.71
13.66*
13.65*
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adenosylcobalamin
(coenzyme B12)
(vitamin B12)
Coenzyme B12 Rearrangements
N
N
O
HO
O
H
P-O
O
O
HN
N N
NN
CONH2
H2NC
O
H2NC
O
H2NC
O
CONH2
CONH2CoIII
OCH2
OH OH
N
NN
N
NH2
a
b
13.73
R
H
O
H
OH
R
H2O
DC
A B
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5-deoxyadenosyl
abbreviation for coenzyme B12
Co
CH2
13.74
R
cobalamin ring
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Conversion of Vitamin B12 to Coenzyme B12
Scheme 13.31
2nd known reaction at C-5 of ATP
Bioynthesis of coenzyme B12
-P3O10-5
H2O
CoIII
CoI
O
OH OH
CH2 AdO
Co
POPO=O3P
O O
O- O-
cob(III)alaminreductase
NADH/FADCoII
cob(II)alaminreductase
13.75
CH2
NADH/FAD
R
adenosylatingenzyme
Mg2+
B12r
B12s
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Scheme 13.32
Light Sensitivity of the Co-C Bond of Coenzyme B12
CH2
Co
R
R
CH2
CoIIh+
13.76 13.77RCH2 is 5'-deoxyadenosyl
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Table 13.1. Coenzyme B12-Dependent Enzyme-Catalyzed Reactions
Enzyme Reaction Catalyzed
CARBON SKELETALREARRANGEMENTS
Methylmalonyl-CoA mutaseCH3
CH COSCoAHOOCHOOCCH2CH2 COSCoA
2-Methyleneglutarate mutase
CH3
CHHOOCHOOCCH2CH2 C COOH
CH2
C COOH
CH2
Glutamate mutase
CH3
CHHOOCHOOCCH2CH2 CH COOH
NH2
CH COOH
NH2
Isobutyryl-CoA mutase CH3
CH COSCoAH3CCH3CH2CH2 COSCoAELIMINATIONS
Diol dehydratase CH CH2OH
OH
R RCH2CHO
R = CH3 or H
Glycerol dehydratase CH CH2OH
OH
HOCH2 HOCH2 CH2CHO
Ethanolamine ammonia lyase CH2 CH2OH
NH2
CH3CHO
ISOMERIZATIONS
L-b-Lysine-5 ,6-aminomutase CH2 CHCH2 COOH
NH2
CH
NH2
H3CCH2 CHCH2 COOH
NH2
CH2H2C
NH2
D-Ornithine-4,5-aminomutase CH2 CH COOH
NH2
CH
NH2
H3CCH2 CH COOH
NH2
CH2H2C
NH2
REDUCTION
Ribonucleotide reductaseO
OH OH
N4-O9P3OO
OH
N4-O9P3O
reductant
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Scheme 13.33
X is alkyl, acyl, or electronegative group
General Form of Coenzyme B12-Dependent Rearrangements
C1
X
C2
H
Y
H
C1 C2 Y
X
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Figure 13.2
Three Examples of Coenzyme B12 Rearrangements
CH2 C
H
H
COOH
CH
NH2
HOOC
CH2 C
H
COOH
CH
NH2
HOOC
H
CH
OH
C
H
H
OHCH3
OH
C
H
OH
O
CH3CH2CH
CH
H
CH3
mutaseglutamate
diol dehydratase
C
H
H
CH2 CH2
NH2
CHCOO-
NH3+
CH2 CHCH2
NH2
CHCOO-
NH3+
H
D-ornithine 4,5-aminomutase
C
A
-H2OB
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Scheme 13.34(1R, 2R) (2S)
No incorporation of solvent protons; therefore no elimination of water (enol would form)
kH/kD = 10-12
Mechanism for Diol Dehydratase and Ethanolamine Ammonia-Lyase
CH3
CHO H
C DHO
H
CH3
C DH
C
14
14
diol dehydratase
13.78 13.79
OH
Stereospecific conversion of (1R,2R)-[1-2H]-[1-14C]propanediol to (2S)-[2-2H]-[1-14C]propionaldehyde catalyzed by diol
dehydratase
Stereospecific [1,2] migration of the pro-R H with inversion
R
R
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Scheme 13.35
S
R
(1R, 2S)
With the (1R, 2S) epimer, the pro-S H migrates; therefore stereochemistry at C-2 determines which C-1 H migrates
Stereospecific Conversion of (1R,2S)-[1-2H]-[1-14C]propanediol to [1-2H]-[1-14C]propionaldehyde Catalyzed by Diol
Dehydratase
14 14
diol dehydrataseCH3
CH OH
C DHO
H
CH3
C HH
COD
13.8113.80
![Page 48: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/48.jpg)
CH3
CH OH
C HRH18O
HS
CH3
CH H
C HHO
H18O
CH3
C HH
CH18O
CH3
CHO H
C HRH18O
HS
CH3
CH H
C HH18O
OH
CH3
C HH
CHO
13.84
- H218O
migrates
13.82
(pro-R hydroxyl group loss)
- H2O
pro-S
pro-R
migrates
13.83
pro-S
pro-R
(pro-R hydroxyl group loss)
HR
HSA
B
Scheme 13.36
(2S)-[1-18O]
(2R)-[1-18O]
The same OH is eliminated (pro-R) regardless of which C-1 H migrates
Stereospecificity of Elimination of WaterDiol dehydratase-catalyzed conversion of (2S)-[1-18O]propanediol to
[18O]propionaldehyde (A) and of (2R)-[1-18O]propanediol to propionaldehyde (B)
Therefore the C-1 H and the C-2 OH migrate from opposite sides giving inversion at both C-1 and C-2
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Scheme 13.37
Crossover Experiment to Show that Diol Dehydratase Catalyzes an Intermolecular Transfer of a Hydrogen from C-1 to C-2
CH3
CH OH
C HHO
3H
H2C
CH2
OH
HO
CH3
CH 3H
CHO
H
CH 3H
CHO
+diol dehydratase
+
13.85
Therefore, hydrogen transfer is intermolecular
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Figure 13.3
Time Course for Incorporation of Tritium from [1-3H]propanediol into the Cobalamin
of Diol Dehydratase
60300
Time (sec)
R a d i o a c t i v i t y
i n C o e n z y m e B 1 2
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OCH2
OH OH
N
NN
N
NH2
Co
aerobic
h
O
H2C
OH OH
N
NN
N
NH2
CoO
C
OH OH
N
NN
N
NH2
OH
O H
+
13.86
anaerobic
h
13.87
Co
OH
+
Scheme 13.38
no 3H here
1/2 3H lost
all 3H retained
no 3H here
Reconstitution of the isolated [3H] coenzyme B12 into apoenzyme with propanediol gives [2-3H]propionaldehyde. All 3H transferred from [3H] coenzyme B12
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
3H here
3H here
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possible intermediate to equilibrate the C-5 protons
13.88 isolated with substrates that cannot rearrange
Synthesized (R,S)-[5-3H] Coenzyme B12 Transfers All 3H to the Product Randomly
O
OHOH
NCH3
13.88
N N
N
NH2
Coenzyme B12 is the hydrogen transfer agent.
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Proposed Rationalization for EPR Spectrum of Co(II) + Carbon Radicals
Scheme 13.39
Formation of 5-deoxyadenosine, cob(II)alamin, and substrate radicals during coenzyme B12-dependent reactions
C
CoIII
CoII
HH
R
C
H
HH
R
+ SubstrateSubstrate-H + Product
![Page 54: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/54.jpg)
Scheme 13.40Not clear if important
Radicals observed in EPR spectrum
Mechanism(s) Proposed for Diol Dehydratase
CH2
Co
RR
CH2H CH
OH
CH
R
CH2
R
CH3
CH
OH
CH
CH3
Co
R
CH3
Co
R
CH3
CH3
OHOH
R
CH2
CH
OH
CH
CH3
13.89
13.90
HO
H
13.91
CH
OH
CH2
CH3
13.92
13.93
HO
C CH2CH3
O
H
CH
OH
CH
CH3
OH13.88
CHHO CH
CH3OHH2O
CoCo
Co
Co
The part shown in the dashed box is even more speculativethan the rest of the mechanism
![Page 55: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/55.jpg)
Scheme 13.41
Chemical Model Study for a Proposed Diol Dehydratase-catalyzed Rearrangement
Involving a Co(III)-olefin -Complex
CH2
Co
N
13CH2 OAcCH2
Co
N
13CH2
CH2
Co
N
13CH2 OMe
CH2
Co
N
13CH2MeO
13.94
13.96
13.97
13.95
MeOH
The trapezoid represents the cobaloxime ligand
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A Cobalt Complex Is Not Necessary
Scheme 13.42
The Fenton reaction as a model for a proposed diol dehydratase-catalyzed free radical rearrangement
HO CH2 CH2 X
HO CH CH2 X
H
HO CH CH2 X HO CH CH2 XH
HO CH CH2
+ H2O2 + Fe2+
HO CH CH2
Fe2+ + H2O2 Fe3+ +
HO CH CH2
+
O CH CH3
CH3CHO
HO
HOH+
-XH
Fe+2Fe+3
![Page 57: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/57.jpg)
(the cobalt complex is just to initiate the reaction by radical generation)
Scheme 13.43
Another Chemical Model Study for a Proposed Diol Dehydratase-catalyzed Free Radical Rearrangement
Co
N
h
OH
OH OH
OH
H
OH
OH
OH
OH
OH
OH
HOH
OH
O
H
or
13.98
13.99
13.100
13.101
-H2O
![Page 58: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/58.jpg)
Scheme 13.44
EPR confirms Co(II) + organic radicalCrystal structures with and without substrates bound show the active site closes upon substrate binding - shields radical intermediates
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
*
CH2
Co
R
DMB
N
NH
610His
CH2
Co
R
N
NH
610His
COO-
HO
SCoAH
H
H
CH3
Co
R
N
NH
610His
COO-
HO
SCoA
H
HCOO-
DMB
H
b
O
SCoA
ba
HH
H
DMB
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.
![Page 59: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/59.jpg)
Figure 13.4
Ab initio calculations disfavor pathway eNo concensus about the others
Six Possible Pathways for the Conversion of Methylmalonyl-CoA Radical to Succinyl-CoA
Radical Catalyzed by Methylmalonyl-CoA Mutase
COO-
HO
SCoA
H
H
COO-
HO
SCoAH
H
COO-
H
H
H
O SCoA
COO-
H
H
H
O SCoA
COO-
HO
SCoAH
H
a COO-
H
H
H
O SCoA
e
d
Co
COO-
HO
SCoAH
H
Co
COO-H
H
H
O SCoA
O
SCoA
COO-
H
H
H
O
SCoA
COO-
H
H
H
H HH COO-
O SCoA
b
c
f
13.10213.103
13.104 13.105
13.106 13.107
13.108
13.109
13.110
13.111 13,112
Co(II)
Co(I)
Co(II)
Co(II)
Co(III)
Co(II)
Co(III)
Co(I)
Co(II)Co(II)
![Page 60: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/60.jpg)
Converts ribonucleotides to deoxyribonucleotidesRibonucleotide Reductase
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)
![Page 61: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/61.jpg)
CH2Ado
Co
S S
CoII
O
OH OH
Ha HbB
B
4-O9P3O
S
S
Ha
S
Ha
S
Ha
SH SHS SH
S SS S
O
O OH
Hb
B4-O9P3OO
O Hb
B4-O9P3O
O
O
HbB4-O9P3O
O
HO H
Ha Hb4-O9P3O
B-BH
BB-
S
Ha
S S
H
O
HO
Hb B4-O9P3O
B-
CH3Ado
•
H
S
S SHB- H H
H
•
•
13.11313.114
13.115
His
13.116
H
13.117
-H2O
C408
C419C119
Scheme 13.45
rates of formation are identical; therefore, concerted reaction
Mechanism Proposed for Coenzyme B12-dependent Ribonucleotide Reductase
![Page 62: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/62.jpg)
Scheme 13.46
regenerates active site for next cycle
reduced by thioredoxin
electrons are transferred to active-site disulfide
The function of the cobalamin in this enzyme is to initiate the radical reaction by abstraction of H• from Cys-408
Mechanism Proposed for Reducing and Reestablishing the Active Site of Coenzyme B12-dependent Ribonucleotide Reductase
CH2Ado
Co
S
CoII
S
SH SHS S B-B-
•
H
13.117
SHSH
H
CH2Ado
SS
His
C408
C731C736 C731C736
C408
C419C119
![Page 63: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/63.jpg)
Figure 13.5
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
FeO
FeO
H2O O
O118His
O
115Glu
O
H2O
O 238Glu
O
204Glu
O
241His
122Tyr
84Asp
H3N CO2
SH3C
O
OH OH
Ade
O
NHH
S
Fe S
FeFe
S Fe
S
OTyr
MnO
Mn
13.118 13.119
13.120
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Scheme 13.47
pro-R
pro-RL--Lys L--Lys
Requires PLP, SAM, [4Fe-4S], and a reducing agent
Reaction Catalyzed by Lysine 2,3-Aminomutase
H3N
NH3+
COO- H3N COO-
H3N
H
HbHaHa
PLP
HHb
13.12213.121
[4Fe-4S]+2SAM
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--LysNo exchange with solvent
![Page 65: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/65.jpg)
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.
C-S bond is stable, unlike C-Co bond
It appears that SAM is functioning like coenzyme B12
In the presence of a reducing agent, [4Fe-4S]+ is observed in the EPR, which reduces SAM to Met and 5-deoxyadenosyl radical
1-6% of 3H ends up in SAM
![Page 66: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/66.jpg)
Ado S COO-
CH3
NH3+
CH2
Ado CH2
H3NNH2
COO-
NH
OH=O3PO
O
H3NN
COO-
HS
NH
OH
CH3
=O3PO
HR
H3N N
COO-
H
NH
OH
CH3
=O3PO
Ado CH3
H3N N
-OOC
H
NH
OH
CH3
=O3PO
Ado CH3
HH H
H
H3N
NCOO-
H
NH
OH
CH3
=O3PO
H
Ado CH2
H
[4Fe-4S]2+
H3N
NCOO-
H
e-
NH
OH
CH3
=O3PO
[4Fe-4S]+
HHAdo CH2
+
Met[4Fe-4S]2+
13.124
13.126
13.127
+ PLP + SAM + [4Fe-4S]+H3N
NH2
COO-
H
Met
H
13.125
H
[4Fe-4S]2+
13.122
13.121
13.123
H2O
Scheme 13.48
not observed in EPR
unique function for PLP
EPR detects organic radicals; 13C label shows product radical 13.126 in EPR spectrum
Mechanism Proposed for Lysine 2,3-Aminomutase
![Page 67: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/67.jpg)
Scheme 13.49
Model Study for New Function of PLPChemical model study to test the proposed rearrangement mechanism for lysine 2,3-aminomutase
CH3
N
CO2Et
Br
Ph
CO2Et
CH3
N
Ph
CH3
N
CO2Et
CH3
Ph
N
Ph
CO2Et CO2Et
CH3
N
Ph
AIBN
-
H SnBu3
Bu3Sn Bu3SnH
Bu3Sn
![Page 68: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/68.jpg)
stabilize -radical
To Get Evidence for Substrate Radical (13.124)
H3NS
NH2
COO-
H
13.128
![Page 69: Chp13.ppt](https://reader035.vdocuments.mx/reader035/viewer/2022062300/55cf8ffc550346703ba2061b/html5/thumbnails/69.jpg)
Scheme 13.50
EPR detected
isolated
Lysine 2,3-aminomutase-catalyzed rearrangement of 4-thialysine to generate a more stable substrate radical
S-
H
Ado–CH3
+
Ado–CH2
+
H3NS
N
COO-
HS
+
HR
– NH3
Ado–CH2
PLP
H
+
Pyr13.129
H3NS
N
COO-
H
H
Pyr
Pyr = pyridine ring of PLP
H3NS
N
COO-
Pyr
13.130
H3NS
N
COO-
Pyr
Ado–CH3
N
COO-
Pyr
H OH
B:
Ado–CH2
N
COO-
Pyr
HO
N
COO-
Pyr13.131
HO
Ado–CH3
COO-
O
H
NH4+
H2O
Evidence for Substrate Radical Formation