Transcript How Do Enzymes Perform and Control Radical Chemistry

How Do Enzymes Perform and Control
Radical Chemistry?
Bernard T Golding
Department of Chemistry
University of Newcastle upon Tyne
Newcastle upon Tyne, UK
Radicals in Enzymatic Reactions
 Radicals are potentially useful intermediates in enzymatic
catalysis because of their high reactivity and special
properties (e.g. ability to cleave non-activated C-H bonds).
 However, reactivity may be towards protein functional
groups and dioxygen.
Hence, the radicals must contain functional groups that
enable tight binding to the protein partner.
Although proteins may be able to shield a bound radical from
dioxygen, radicals are primarily found as intermediates
with anaerobic organisms.
(W Buckel and B T Golding, FEMS Microbiol Revs, 1999, 22, 523-541)
Examples of Radicals in Enzymatic Reactions
• Coenzyme B12-dependent enzymatic reactions
• Ribonucleotide reductases (e.g. human enzyme and
Escherichia coli)
• a-Lysine 2,3-aminomutase (‘poor man’s B12)
• Cytochrome P-450 dependent monooxygenases
• Penicillin biosynthesis
• Pyruvate formate lyase
and many more!
Coenzyme B12-dependent
Enzymatic Rearrangements
H X
a
C C
X H
a
b
Y H
ENZYME
Diol dehydratase
Ethanolamine ammonia lyase
Methylmalonyl CoA mutase
Glutamate mutase
2-Methyleneglutarate mutase
C C
Y H
a
H or Me
H
H
H
H
b
H, Me, CF3
H or Me
H
H
H
X
OH
NH2
COSCoA
CH(NH3+)CO2C(=CH2)CO2-
Y
OH
OH
CO2H
CO2H
CO2H
b
The Carbon Skeleton Mutases:
Glutamate Mutase
 This enzyme was first isolated from the anaerobic
bacterium Clostridium tetanomorphum and catalyses the
rearrangement of glutamate to 3-methylaspartate:
H2N
O2C
H
S
Hpro-S
CO2
H
O2C
CO2
H S S
H
H2C
NH2
H
H A Barker found that the enzyme contained a light-sensitive,
yellow-orange cofactor, which was subsequently identified as
coenzyme B12.
(review: W Buckel and B T Golding, Chem Soc Revs, 1996, 26, 329-337)
Structure of Coenzyme B12
CONH2
CONH2
Me
b
Me
c
Me
H2NOC
A
a
N
Me
R
B
d
HO
N
N
H2NOC
f
R (5'-deoxyadenosyl)
N
D
g
C
e
Me Me
NH
-
O P
O
O
N
N
N
Me
Ado
H
H
Me
Co
HO
Me
O
Me CONH2
N
O
= HC
2
Me
N
O
OH
N
Co
H
CONH2
O
CH2OH
adenosylcobalamin
AdoCH2-Cbl
NH2
Stereochemistry of Glutamate Mutase
• Hpro-S is abstracted from C-4 of glutamate.
• The abstracted H mixes with the 5'-methylene hydrogens
of adenosylcobalamin.
• The glycinyl residue migrates to this C-4 with inversion of
configuration.
H2N
O2C
H
S
O2C
CO2
S
S
H
H
H2C
NH2
H
CO2
H
Hpro-S
Ado
H
H
Co
adenosylcobalamin
Reaction Pathway for Glutamate Mutase
Binding of the substrate to the enzyme-coenzyme complex
triggers Co-C bond homolysis:
Ado
H
Ado
H
H
H
Co
Co
The adenosyl radical initiates the reaction pathway by hydrogen
atom abstraction from a substrate molecule:
-
O2C
H
H
Hpro-S
AdoCH2
NH2
S
CO2-
H
-
O2C
H2N
NH2
CO2
-
CO2-
H
H
AdoCH3
S
CH2
-
H
CO2-
H2N
-
O2C
O2C
AdoCH3
S
CH2
H
AdoCH2
Possible Rearrangement Mechanisms for
Glutamate Mutase
Fragmentation-recombination pathway:
H
-
O2C
H2N
NH2
CO2
-
H
-
H
O2C
CO2
-
H2N
CO2-
H
H
-
H
O2C
H
Note that this mechanism has strict stereoelectronic requirements:
the s-bond undergoing cleavage must be properly aligned with the
p-orbital of the 4-glutamyl radical.
Possible Rearrangement Mechanisms for
Glutamate Mutase
Addition-elimination via an intermediate imine:
-
O2C
H
H
NH2
CO2-
-
O2C
H
H
H
CO2
-
N
-
CO2
N
-
CO2-
O2C
H
H
X
N
O2C
X
X
H
X
-
H
N
CO2
X
CO2H
-
N
H2N
CO2
H
CH2
-
O2C
-
O2C
-
CH3
-
O2C
X contains a carbonyl group from the protein or a cofactor
(e.g. pyridoxal)
Tools for Elucidating the Mechanism of
Coenzyme B12-dependent Reactions
 Synthesis of substrate analogues, including isotopically
labelled compounds.
 NMR and EPR studies of enzymatic reactions using
substrate analogues.
 Model studies.
 Ab initio calculations of reaction pathways (with
Professor Leo Radom, Canberra).
EPR Study of Glutamate Mutase
• Glutamates specifically labelled with 2H, 13C and 15N were
purchased/synthesised.
• Each compound was incubated with glutamate mutase +
coenzyme B12 for ca. 20 s.
• The reaction mixtures were frozen in liquid N2 and EPR
spectra obtained.
• These experiments identified the 4-glutamyl radical as an
intermediate:
H
-
O2C
H
NH2
CO2-
EPR Study of Glutamate Mutase
A) [4-13C]-(S)-glutamate.
B) [3-13C]-(S)-glutamate.
C) [2-13C]-(S)-glutamate.
D) unlabelled (S)-glutamate.
(All spectra were recorded at 50 K)
X
280
300
320
340
360
380
Field (mT)
EPR spectra of the radical species derived from incubating glutamate
mutase and coenzyme B12 with 13C-labelled (S)-glutamate.
2-Methyleneglutarate Mutase
2 AdoCH2-Cbl
• 2-Methyleneglutarate mutase from Clostridium barkeri
catalyses the equilibration of 2-methyleneglutarate with
(R)-3-methylitaconate:
-
O2C
-
H
O2C
Hpro-R
K = 0.06
-
H
O2C
CO2
-
Me
The pink-orange enzyme is a homotetramer (300 kDa) containing AdoCH2-Cbl.
Removal of the coenzyme gives inactive apoenzyme, which can be re-activated
by addition of AdoCH2-Cbl.
The active enzyme is susceptible to dioxygen, which converts bound
AdoCH2-Cbl into hydroxocobalamin.
(C Michel, S P J Albracht, and W Buckel, Eur J Biochem, 1992, 205, 767)
Addition-elimination Mechanism for the
Rearrangement
Equilibration of 2-methyleneglutarate 1a and (R)-3-methylitaconate
2a and their corresponding radicals 3 and 4 via cyclopropylcarbinyl
radical 5:
-
O2C
H
-
-
O2C
1a
O2C
Me
H
H
-
O2C
H
.
-
O2C
.
H
-
O2C
-
O2C
3
CO2-
H
R
5
H
-
H
CO2
O2C
H
2a
R
.
H
4
-
Test of the Cyclopropylcarbinyl Mechanism
• If the energy barrier to rotation about the C-1/methylene bond in
the cyclopropylcarbinyl radical is sufficiently low, then a
stereospecifically deuteriated 3-methylitaconate (say the Zisomer 2b) should equilibrate with its E-isomer 2c when
incubated with 2-methyleneglutarate mutase holoenzyme.
-
CO2-
H
O2C
Z
Me
D
-
CO2-
H
O2C
2b
E
Me
H
H
2c
D
H
-
O2C
?? via
-
H
.
D
1
O2C
It does and also equilibrates with the corresponding E and Z isomers
of 2-methyleneglutarate.
Do These Results Prove the
Cyclopropylcarbinyl Mechanism?
• Consider an alternative mechanism (‘fragmentationrecombination’) in which the substrate-derived radical 3 fragments
to acrylate and the 2-acrylate radical 6 (path b).
-
O2C
-
H
-
1a
O2C
O2C
Me
H
H
-
O2C
H
.
-
path a
O2C
.H
-
H
-
O2C
-
3
5
2a
R
H
CO2
O2C
H
O2C
CO2-
H
-
.
H
4
path b
-
-
O2C
.
6
H
H
O2C
H
A rotation within the acrylate
radical can explain the NMR results
Can The Two Mechanisms Be Distinguished?
• For conversion to the
cyclopropylcarbinyl radical, the
conformation shown is essential to
achieve maximal overlap between the
p orbitals at C-2 and C-4.
Position of this
group can differ
in the two pathways
Critical bond
H
-
-
• For the fragmentation pathway, it
suffices to achieve maximal overlap
between the p orbital at C-4 and the
critical C-2/C-3 s-bond.
• The two alternative mechanisms can in
principle be distinguished by the
conformation of the substrate bound to
the enzyme.
O2C
O2C
path a
2
H
H
-
4
O2C
3
- R
O2C
D
-
path b
O2C
H
-
O2C
D
H
H
D
Methylmalonyl-CoA Mutase
This human enzyme converts the (R)-isomer of
methylmalonyl-CoA to succinyl-CoA (RS = coenzyme A):
O
O
H
RS
HO2C
R
CH2
from propionate, a
toxic product of the
degradation of fats
HO2C
H
SR
pro-R
H
enters Krebs cycle
Stereochemistry of Methylmalonyl-CoA Mutase
• In contrast to glutamate and 2-methyleneglutarate mutase,
the migrating group (thioester residue) migrates with
retention of configuration at the receiving locus:
H
H
O2C
O2C
Hpro-R
O
SR
R
O
CH2
H
SR
Can this result be explained on mechanistic grounds?
Pathways for Methylmalonyl-CoA Mutase
• Consider three possible mechanisms for the
interconversion of intermediate radicals, corresponding in
structure to substrate and product:
Fragmentation-recombination:
O
O
C SCoA
H2C
H
CO2
Radical corresponding to
methylmalonyl-CoA
SCoA
C SCoA
O
CO2
-
H
CO2-
Radical corresponding to
succinyl-CoA
Pathways for Methylmalonyl-CoA Mutase
Addition-elimination:
.O
O
.
HC
SCoA
C SCoA
2
SCoA
.
O
H
CO2-
H
CO2-
H
CO2-
Addition-elimination after protonation:
HO
.
HC
2
HO
C SCoA
H
CO2
SCoA
.
SCoA
HO
H
CO2
.
H
CO2-
Mechanisms for the Rearrangement of the (R)Methylmalonyl Radical to the Succinyl Radical
.
.
O
RS
RS
HO2C
RS
O
SR
HO2C
HO2C
TS
O
.
O
RS
.
O
TS
.
Re
face
O
.
HO2C
HO2C
HO2C
H
TS
.
+H
OH
RS
HO2C
RS
.
-H
OH
HO
HO2C
HO2C
TS
(RS = coenzyme A)
.
SR
SR
Calculation of Reaction Pathways
• Ab initio molecular orbital calculations were carried out on
a model reaction, the degenerate rearrangement of the 3propanal radical:
O
.
O
C H
H2C
H
intermediate
radical
H C
2
H
H
.CH
H
Pathway
#
-1
H (kJ mol )
Fragmentationrecombination
96
Addition-elimination
47
Addition-elimination after
protonation
10
(cf. D. M. Smith,, B. T. Golding, and L. Radom, J. Am. Chem. Soc., 1999, 121, 1037 and 1383)
Possible Mechanisms for the Degenerate
Rearrangement of the 3-Propanal Radical
.
.
O
TS
O
.
O
O
.
O
TS
.
.
TS
.
+H
OH
.
OH
TS
-H
.
HO
O
How Can Protonation be Tapped?
 The pKa of the thioester group of methylmalonyl-CoA or
succinyl-CoA is ca. - 6.
• Even the strongest conceivable acid in a protein cannot
generate a significant concentration of protonated
carbonyl.
H X
• Can partial protonation by a
weaker acid (H-X) help?
O
C SCoA
H2C
H
CO2
Quantifying Partial Protonation
The effect of protonating the 3-propanal radical by three
different acids was investigated using MO theory:
Acid
Proton affinity C=O……HX C=O distance Rearrangement
of conjugate
distance (Å)
(Å)
base (kJ mol-1)
None
energy barrier
(kJ mol-1)

1.209
46.9
HF
1556
1.727
1.221
41.4
NH4+
849
1.503
1.235
24.5
H3O+
680
1.046
1.273
10.3
0.976
1.299
10.0
Full protonation
Why Does Partial Protonation Help?
• The lowering of the reaction barrier by protonation is due
to the stronger interaction of the transition state with the
proton.
• Even a small amount of proton transfer to C=O results in a
significant decrease in the barrier, e.g. with HF [which
models a glutamic or aspartic acid carboxyl group in a
protein (n.b. PA of formate = 1431 kJ mol-1)].
• With NH4+, which models protonated lysine or histidine in
a protein, the lowering of the barrier corresponds to a rate
increase of ca. 105.
Partial Protonation and Hydrogen Bonding
• Enzymes often anchor their substrates by hydrogen
bonding, e.g. the carbonyl group of methylmalonyl-CoA is
hydrogen bonded to HisA244 in the mutase:
Proposal: Any reaction that is facilitated by protonation will be
facilitated by the partial protonation that hydrogen bonding
provides.
• Enzymes may utilise hydrogen bonding for binding and catalysis.
Active Site of Methylmalonyl-CoA Mutase
Substrate
His244
Nearest histidine N - substrate C=O
separation = 2.95 Å
Tyr89
Cobalt - substrate C=O
separation = 8.5 Å
Corrin
Arg207
(F Mancia and P R Evans,
Structure, 1998, 6, 711)
Possible Rationalisations for
(a) the Inversion Pathway of Glutamate Mutase
(b) the Retention Pathway of Methylmalonyl-CoA Mutase
Re face
a
-
O2C
H
H
NH2
S
CO2-
-
O2C
.
H
NH2
CO2-
.
-
.
CO2
H2N
H
-
O2C
S
.
O2C
AdoCH3
CH2
H
AdoCH2
b
.
-
O2C
H
AdoCH3
O
SR
-
O2C
pro-R
H
.
Re face
AdoCH2
H
.
O
O
SR
RS
AdoCH3
.
RS
CH2
-
O2C
-
S
CH2
AdoCH3
AdoCH2
CO2
H
-
H
Hpro-S
H2N
-
O2C
AdoCH2
O
H
R
CH2
In path b, migration to the Re face may be blocked by deoxyadenosine.
Current Status of Mechanisms for the
Carbon Skeleton Mutases
• For glutamate mutase, fragmentation-recombination may
be the only possibility.
• For 2-methyleneglutarate mutase, addition-elimination or
fragmentation-recombination remain as possibilities.
• Addition-elimination facilitated by partial protonation is
highly plausible for methylmalonyl-CoA mutase.
Note that all of these pathways are energetically permissible, i.e. they have barriers
below the highest energy barrier in the overall pathway, which is for H atom
abstraction steps (estimated at 60-75 kJ mol-1 for methylmalonyl-CoA mutase).
Acknowledgements
• Daniele Ciceri, Anna Croft, Dan Darley,
Ruben Fernandez, Joachim Winter (Newcastle)
• Wolfgang Buckel, Harald Bothe, Gerd Bröker,
Antonio Pierik (Marburg)
• Leo Radom and David Smith (Canberra)
• European Commission