chp12
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studyyTRANSCRIPT
The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 12
Formylations, Hydroxymethylations, and
Methylations
Transfer of one-carbon unitscan be oligomer of up to 12 Glu residues
tetrahydrofolatenamed as polyglutamate derivatives of tetrahydrofolate (H4PteGlun) [Pte - pteroate]
Tetrahydrofolate-dependent Enzymes
HN
N
NH
HN
NH
C
O
CHCH2CH2COO-
COO-
H2N
O
H6
glutamate5,6,7,8-tetrahydro-pteridine
pKa of acid is -1.25pKa of acid = 4.8
p-amino-benzoic acid
10
12.1
HN
5
5
10
abbreviated structure for tetrahydrofolate
12.2
HN
N
NH
HNH2N
O NHR'
pteroate ring
Folic Acid (a vitamin for humans)
HN
N
N
N
NH
CNHO
CHCH2CH2COO-
COO-
H2N
O
12.3
Reduction of Folate to Tetrahydrofolate
Scheme 12.1
dihydrofolate
Reactions catalyzed by dihydrofolate reductase (DHFR)
+BH
N
H H
NH2
O
R N
NH2
OH
R
NR
H O
NH2
B+
H
HN
N
N
N
NHR'
H2N
O
HN
N
N
HN
NHR'
H2N
O
HN
N
N
HN
NHR'
H2N
O
HN
N
NH
HN
NHR'
H2N
O
H
dihydrofolatereductase
+
12.1
..
12.3
+
N
H H
NH2
O
R
dihydrofolatereductase..
Lys
HN+
NH
OH
:NH2COO-
H
OH
HN+
NH
OH
OHB:
O CH2
HN+
NH
OH
HN+
NH
OH
HN+
NH
OH
COO-H
COO-
+B
COO- H
H
H
HN+
NH
OH
COO-HH Lys
:NH2
Lys
HN+
NH
OHH2N COO-
COO-H
+
=O3PO
=O3PO=O3PO=O3PO =O3PO
=O3PO =O3PO
*
*
*
+ +
++
See Scheme 8.39
..
+
12.4
Scheme 12.2
-cleavage
pro-S hydrogen added/removed
(retention of configuration)
Ordered mechanism: no conversion of Ser to H2C=O unless tetrahydrofolate is bound
Generation of the Transferring Carbon UnitSerine hydroxymethyltransferase-catalyzed formation of formaldehyde via a proposed -cleavage mechanism. The asterisk indicates the carbon unit that becomes the one-carbon unit transferred in tetrahydrofolate-dependent enzymes.
The one-carbon unit can be transferred in 3 oxidation states
Scheme 12.3
N5-methylene H4Pte
N5,N10-methylene H4Pte
N10-methylene H4PteKeq = 3.2 x 104 in favor of 12.7
Transfer of One-Carbon Units
Serine hydroxymethyltransferase-catalyzed reaction of formaldehyde and tetrahydrofolate to methylenetetrahydrofolate
Transfer at the formaldehyde oxidation state (transfer HOCH2
- group)
HN
N
NH
HNH2N
O NHR'
HN
B+ H
O CH
H
N
N
HNH2N
O NHR'HCH2
OHB:+B
H
N
CH2
HN
NHR'O
H2NHNN
HN
N
N
HNH2N
O NR'CH2
:B
HB H
NR'CH2
O
H2NHN
NH
N
HN+
** +
:*
+
*
*
..
12.5
12.6
12.7
12.8
5
10
-H2O
Scheme 12.5
N5,N10-methenyltetrahydrofolate cyclohydrolase
N10-formyl H4PteN5-formyl H4Pte
Transfer at the Formate Oxidation State (transfer formyl group) Oxidation of N5,N10-methylenetetrahydrofolate to N5,N10-
methenyltetrahydrofolate catalyzed by methylenetetrahydrofolate dehydrogenase and hydrolysis of N5,N10-methenyltetrahydrofolate to
N5,N10-methenyltetrahydrofolate cyclohydrolase
NHN
NHNH2N
O NR'H
H
N
NH2
O
N
R
NR'
H
O
H2NHNN
HN
NHN
NHNH2N
O NR'
H O
:B
HO H
N
R
O
NH2
HH
H
:B
B+H
+BH
NR'O
H2NHN
NH
N
HN
H
OH
HN
N
N
HNH2N
O NHR'
Ob
*
+
*
*+
..
12.9
a*
b a
12.1212.11
12.10
*
N5-methyl H4Pte
Requires NADPH and FAD to make 12.13 from N5-methylene H4Pte
Transfer at the Methanol Oxidation State (transfer methyl group)
N
CH3 NHR'O
H2NHNN
HN
12.13
H5 6
Excludes [1,3]-hydride shift
Excludes tautomerization of N5-methylene H4Pte to 12.14
N
CH3NHR'O
H2NHNN
HN
12.14
N
CH2NHR'O
H2NHNN
HNH
N
CH2NHR'O
H2NHNN
HN
3H
Reaction Run Backwards with [6-3H]-12.13 Releases No 3H and Does Not Transfer 3H to Methyl
Scheme 12.7
Proposed Mechanism for the Reduction of N5,N10-methylenetetrahydrofolate by
N5,N10-methylenetetrahydrofolate Reductase
FAD
B
FADH-
BH
N
CH2
HN
NHR'O
H2NHNN
HN
N
N
HNH2N
O NHR'H2C
FADH-
BN
CH3 NHR'O
H2NHNN
HN
FAD
B
NADPH +H+ NADP+
+
+
+ N
CH3 NHR'O
H2NHNN
HN
+
12.13
12.6 12.7
Scheme 12.8
With [5-3H]-deazaFADH2, 3H transferred to
methyl group, consistent with this mechanism
Proposed Alternative Hydride Mechanism for N5,N10-Methylenetetrahydrofolate Reductase
12.7
FAD FADN
CH2
HN
NHR'O
H2NHNN
HN
N
N
HNH2N
O NHR'H2C
D-NADPH +D+ NADP+
+
+
12.6
N
CH2 NHR'O
H2NHNN
HN
D
D2O
Proposed mechanism for glycinamide ribonucleotide (GAR) transformylase
Scheme 12.9GAR FGAR
Transfer of a Formyl Group
O
=O3PO
HO OH
HNNH2
O
NR'O
H2NHN
NH
N
HN
HO
O
=O3PO
HO OH
HNN
O
NR'O
H2NHN
NH
N
HN
H O
B
H
H
H
B: O
=O3PO
HO OH
HNNH
O
12.16
NHR'O
H2NHN
NH
N
HN
H O
12.17
HB
12.2
H
12.18
:B
..
Third step in biosynthesis of purines
O
HO
HN
N=O3PO
O
H
HO
N
N
N
HN
N
H2N
HO
H
R
O
HO
HN
N=O3PO
O
CH3
HO
N
N
N
HN
NH
R
H2N
HO
12.19
++
12.20
5
6
Transfer at Formaldehyde Oxidation State
Scheme 12.10
exchanges reduced(normally CH2OH)
oxidized
C-5 H exchanges with solvent
Reaction catalyzed by thymidylate synthase (an anomalous transfer of a methylene group)
Last step in de novo biosynthesis of thymidylate
inverse 2° isotope effect rehybridization of C-5 from sp2 sp3
inverse 2° isotope effect at C-6 also
Scheme 12.11
transferred to
Transfer of the C-6 Hydrogen of N5,N10-Methylenetetrahydrofolate to the Methyl Group of Thymidylate Catalyzed by Thymidylate Synthase
dRP
HN
N
O
H
HOHN
N
N
HN
N
H2N
O
3H
R dRP
HN
N
O
C3HH2
HO
HN
N
N
HN
NH
R
H2N
O
++
Me
MeN
N
O
D
HOMe
MeN
N
O
D
HOOH
H OH
Me
MeN
N
O
D
HOOH
H
Me
MeN
N
O
HOOH
H
Me
MeN
N
O
H
HO
12.21 12.22OH
OH
C-5 2H washed out in base
Scheme 12.12note: C-5 and C-6 are rehybridized to sp3
Thiols are more effective than hydroxide
Chemical Model Study for Thymidylate Synthase-catalyzed Exchange of the C-5
Hydrogen of 2-Deoxyuridine-5-monophosphate
Scheme 12.13structure identified
by X-ray
Inactivation of Thymidylate Synthase by 5-Fluoro-2-deoxyuridylate
MeN
N
O
F
HOO
HO
HN
N=O3PO
O
CH2
SO
Cys
FN
NH
N
HN
H2N
NH
C
Glu
O
O
12.24
O
HO
=O3PO
12.23
Scheme 12.14
Proposed Mechanism for the First Part of the Reaction Catalyzed by Thymidylate Synthase Based on
Inactivation Complex with 5-Fluoro-2-deoxyuridylate
O
HO
HN
N=O3PO
O
HO
H
SCys
HN
N
N
HN
CH2 NHR
H2N
O
H
O
HO
HN
N=O3PO
O
HO
H
SCys
HN
N
N
HN
H2C NR
H2N
O
HHN
N
N
HN
CH2NHR
H2N
O
H
O
HO
HN
N=O3PO
O
HO
H
SCys
BH B
..
B
Original Proposal
Scheme 12.15
[1,3]-H shift suprafacial
Not allowed by Woodward-Hoffman rulesShould have occurred with 5-F analogue, but does not
Highly unlikely [1,3]-H shift mechanism for reduction of the substrate catalyzed by thymidylate synthase
HN
N
N
HN
CH2 NHR
H2N
O
H
O
HO
HN
N=O3PO
O
HO
H
SCys
B
12.25
HN
N
N
HN
CH3NHR
H2N
O
O
HO
HN
N=O3PO
O
HO
H
SCys
..
To Rationalize Stability of 5-F Adduct
Scheme 12.16
Proposed mechanism for the second part of the reaction catalyzed by thymidylate synthase
HN
N
N
HN
CH2 NHR
H2N
O
H
O
HO
HN
N=O3PO
O
HO
H
SCys
B
HN
N
N
HN
CH2 NHR
H2N
O
H
O
HO
HN
N=O3PO
O
HOS
Cys
BH
HN
N
NH
HN
CH2
NHR
H2N
O
H
O
HO
HN
N=O3PO
O
HOS
Cys
CH3
O
HO
HN
N=O3PO
O
HOS
Cys
HN
N
N
HN
NHR
H2N
O
CH3
O
HO
HN
N=O3PO
O
HO
12.25
SCys
12.26
:
..
when F, it is stable
Precedence for Elimination Mechanism
Scheme 12.17
Model study for the formation of the C-5 exo-methylene intermediate proposed in the reaction catalyzed by thymidylate synthase
Me
MeN
N
O
CH2
HO
NO2O
Me
MeN
N
O
CH2
HO
NO2O
OH Me
MeN
N
O
CH2
HOOH
Me
MeN
N
O
CH2OH
HOOH
Me
MeN
N
O
CH2OH
HO
12.27
OH
OH
Enzymatic Intermediate Trapped with -Mercaptoethanol
Scheme 12.18isolated
Trapping of the proposed C-5 exo-methylene intermediate during catalytic turnover of thymidylate synthase
O
HO
HN
N=O3PO
O
HOS
Cys O
HO
HN
N=O3PO
O
HOS
Cys O
HO
HN
N=O3PO
O
HO
12.28
SCys
12.26
SOH S
OHS
OH
Scheme 12.19
Alternative proposed electron transfer mechanism for the reduction of the exo-methylene intermediate in the reaction catalyzed by thymidylate synthase
Alternative to Hydride Transfer
12.29
HN
N
NH
HN
CH2
NHR
H2N
O
H
O
HO
HN
N=O3PO
O
HOS
Cys
HN
N
NH
HN
CH2
NHR
H2N
O
H
O
HO
HN
N=O3PO
HO
SET SET
12.30
SCys
O
HN
N
N
HN
CH3
NHR
H2N
O
O
HO
HN
N=O3PO
HOS
Cys
OH
HN
N
N
HN
NHR
H2N
O
CH3
O
HO
HN
N=O3PO
HOS
Cys
O
CH3
O
HO
HN
N=O3PO
O
HOS
Cys
12.26
-H+
Transfer at the Methanol Oxidation State
Scheme 12.20
Reaction catalyzed by the cobalamin-independent methionine synthase
N
HNH2N
O
HN
N
CH3 NHR
HS COO-
NH3+
NH
HNH2N
O
HN
NCH3S
NHR
COO-
NH3+
+
12.31
+
12.32
Two different forms of methionine synthase:• one transfers CH3 directly from N5-methyl H4PteGlu
• one first transfers CH3 to a cobalt complex (cobalamin)
Scheme 12.21
SN2
increases leaving group ability (model for protonated N5-Me-H4PteGlu)
Enzyme requires Zn2+ (coordinates to the thiol S)
Model Study for the Reaction Catalyzed by the Cobalamin-independent Methionine Synthase
N
HN
HN
O
HN
N
CH3
CH3
S COO-
NH3+
O
CH3
H3C
12.33
+N
HN
HN
O
HN
N
CH3
CH3O
CH3 CH3S COO-
NH3+
+Na+
corrin ring
methylcobalaminfrom the methylation of cob(I)alamin by N5-MeH4PteGlu
Cobalamin-dependent Methionine Synthase
N
NH3C
H3C
OHO
O
CH2OHHP-OO
O
HN
CH3N
CH3
N
CONH2
NN
H3C
H3CH2NOC
H3CH3C
H2NOCCH3
CONH2
CH3
CONH2
H3C
CH3
CONH2
12.34
H
H
O
C
A
Co+
B
D
Scheme 12.22
retention of Me configuration
Cleland notation
(A) Reaction Catalyzed by Cobalamin-dependent Methionine Synthase
(B) Cleland Diagram for the Reaction Catalyzed by Cobalamin-dependent Methionine Synthase
E cob(I)alamin
CH3-H4PteGlu
E CH3-cobalamin
HCys Met
E cobalamin
HCys
E CH3-cob(I)alamin
Met
E cobalamin
H4PteGlu
H4PteGluCH3-H4PteGlu
A
B Enzyme reactionpathway
Scheme 12.23
Model Study for the Methylation of Cob(I)alamin during the Reaction Catalyzed by the
Cobalamin-dependent Methionine Synthase
N
HN
HN
O
HN
N
CH3
CH3O
CH3
H3C
12.33
N
HN
HN
O
HN
N
CH3
CH3O
CH3
cob(I)alamin methylcobalamin
12.35
O
HO
CH2 A
OH
O
O
HO
A
OH
S-OOC
H3N
CH3P
O
O-
OP
O
O-
O=O3P
12.36
CH3S
COO-
H3N
PPi + Pi
methionineadenosyltransferase
S-Adenosylmethionine (SAM)-DependentTransfer of CH3
Scheme 12.24
rare attack at C-5
ATP SAM
more common methylating agent
Proposed mechanism for the synthesis of S-adenosylmethionine catalyzed by methionine adenosyltransferase
Scheme 12.25
With chiral CH3 group gives inversion of stereochemistry
Generalized Reaction Catalyzed by S-adenosylmethionine-dependent
Methyltransferases
O
HO
A
OH
S-OOC
H3N
CH3
XRH :B
XRCH3 O
HO
A
OH
S-OOC
H3N
12.36
+
12.37
SN2
Scheme 12.26
indolylpyruvate indolmycin
inversion of stereochemistry
Stereochemistry of Methylation of Indolylpyruvate in the Biosynthesis of Indolmycin
NH
O
COO-
HH
:B
NH
O
COO-
H
S
adenosyl
COO-
H3N D T
H
NH
O
COO-
HD
TH
NH
H
DT
H
N
O
H
12.39
B:
12.38
O
NHCH3