the organic chemistry of enzyme-catalyzed reactions chapter 13 rearrangements
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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
Scheme 13.1
[3,3] sigmatropic rearrangement
General form of the Claisen rearrangement
O O
H
Sigmatropic Rearrangements
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
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‡
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
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+
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
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
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
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
hapten to raise the antibody
OH
O
NH
ONH2
13.12
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
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)
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
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
‡
Hapten used
N
NHO
O
NHAc
O
O
-OOCH
H
13.20
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
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
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
substrate
CH3 C
OH
C
O
COO-
CH3
13.36
Kinetically-competent intermediate
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
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
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
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
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
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
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
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
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
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
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
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
HN
N N
NH
O O
O O
DNA photolyase
hν (uv)
hν (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
hν (uv)
hν (visible)
13.64
DNA Photolyase UV light causes DNA damage
Reactions catalyzed by DNA photolyase and (6-4) photolyase
Scheme 13.27
visible hν used as a substrate for photoreactivationcyclobutane pyrimidine dimer
(6-4) photoproduct
both types carcinogenic, mutagenic
Rearrangements Via Radical Intermediates
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
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
*hν (300-500 nm)
13.65
*
P
13.63
13.68
13.69 13.70 13.71
13.66*
13.65*
Scheme 13.29
Proposed Mechanism for the Formation of the (6-4) Photoproduct
HN
NO
O
P
OH
H N
N O
13.72
hν (uv)
13.64
HN
NO
O
P
O
H N
N O
HHN
NO
O
P
O
H N
N O
H
[2+2]
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
*hν (300-500 nm)
13.65
HN
NO
O
13.72
13.64
P
13.66
O
H N
N OH
*
13.71
13.66*
13.65*
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
5-deoxyadenosyl
abbreviation for coenzyme B12
Co
CH2
13.74
R
cobalamin ring
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
Scheme 13.32
Light Sensitivity of the Co-C Bond of Coenzyme B12
CH2
Co
R
R
CH2
CoIIhν+
13.76 13.77RCH2 is 5'-deoxyadenosyl
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-O9P3Oreductant
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
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
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
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
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
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
Figure 13.3
Time Course for Incorporation of Tritium from [1-3H]propanediol into the Cobalamin
of Diol Dehydratase
60300
Time (sec)
OCH2
OH OH
N
NN
N
NH2
Co
aerobic
hν
O
H2C
OH OH
N
NN
N
NH2
Co OC
OH OH
N
NN
N
NH2
OH
O H
+
13.86
anaerobic
hν
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
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.
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
CoIIICoII
HH
R
C
H
HH
R
+ SubstrateSubstrate-H + Product
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
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
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
(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
hν
OH
OH OH
OH
H
OH
OH
OH
OH
OH
OH
HOH
OH
O
H
or
13.98
13.99
13.100
13.101
Δ
-H2O
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.
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)
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)
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
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
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
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
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
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
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
AIBNΔ
-
H SnBu3
Bu3Sn Bu3SnH
Bu3Sn
stabilize -radical
To Get Evidence for Substrate Radical (13.124)
H3NS
NH2
COO-
H
13.128
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
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