How Do Enzymes Perform and Control Radical Chemistry? Bernard T Golding Department of Chemistry University of Newcastle upon Tyne Newcastle upon Tyne, UK - PowerPoint PPT Presentation

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How Do Enzymes Perform and Control Radical Chemistry? Bernard T Golding Department of Chemistry University of Newcastle upon Tyne Newcastle upon Tyne, UK

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Pyruvate formate lyase. and many more! Coenzyme B12-dependent. Enzymatic Rearrangements ... acid carboxyl group in a protein (n.b. PA of formate = 1431 kJ mol-1) ... – PowerPoint PPT presentation

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Title: How Do Enzymes Perform and Control Radical Chemistry? Bernard T Golding Department of Chemistry University of Newcastle upon Tyne Newcastle upon Tyne, UK


1
How Do Enzymes Perform and Control Radical
Chemistry? Bernard T Golding Department of
Chemistry University of Newcastle upon
Tyne Newcastle upon Tyne, UK
2
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)

3
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 mans B12)
  • Cytochrome P-450 dependent monooxygenases
  • Penicillin biosynthesis
  • Pyruvate formate lyase
  • and many more!

4
Coenzyme B12-dependent Enzymatic Rearrangements
5
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

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)
6
Structure of Coenzyme B12
adenosylcobalamin AdoCH2-Cbl
7
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.

8
Reaction Pathway for Glutamate Mutase
Binding of the substrate to the enzyme-coenzyme
complex triggers Co-C bond homolysis
The adenosyl radical initiates the reaction
pathway by hydrogen atom abstraction from a
substrate molecule
9
Possible Rearrangement Mechanisms for Glutamate
Mutase
  • Fragmentation-recombination pathway


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.
10
Possible Rearrangement Mechanisms for Glutamate
Mutase
Addition-elimination via an intermediate imine
X contains a carbonyl group from the protein or a
cofactor (e.g. pyridoxal)
11
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).

12
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

13
EPR Study of Glutamate Mutase
A) 4-13C-(S)-glutamate. B) 3-13C-(S)-glutamat
e. C) 2-13C-(S)-glutamate. D) unlabelled
(S)-glutamate. (All spectra were recorded at 50
K)
EPR spectra of the radical species derived from
incubating glutamate mutase and coenzyme B12
with 13C-labelled (S)-glutamate.
14
2-Methyleneglutarate Mutase
  • 2-Methyleneglutarate mutase from Clostridium
    barkeri catalyses the equilibration of
    2-methyleneglutarate with (R)-3-methylitaconate
  • 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)
15
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
16
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 Z-isomer 2b) should equilibrate with its
    E-isomer 2c when incubated with
    2-methyleneglutarate mutase holoenzyme.

It does and also equilibrates with the
corresponding E and Z isomers of
2-methyleneglutarate.
17
Do These Results Prove the Cyclopropylcarbinyl
Mechanism?
  • Consider an alternative mechanism
    (fragmentation-recombination) in which the
    substrate-derived radical 3 fragments to acrylate
    and the 2-acrylate radical 6 (path b).

A rotation within the acrylate radical can
explain the NMR results
18
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.
  • 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.

19
Methylmalonyl-CoA Mutase
This human enzyme converts the (R)-isomer of
methylmalonyl-CoA to succinyl-CoA (RS coenzyme
A)
20
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

Can this result be explained on mechanistic
grounds?
21
Pathways for Methylmalonyl-CoA Mutase
  • Consider three possible mechanisms for the
    interconversion of intermediate radicals,
    corresponding in structure to substrate and
    product
  • Fragmentation-recombination

Radical corresponding to methylmalonyl-CoA
Radical corresponding to succinyl-CoA
22
Pathways for Methylmalonyl-CoA Mutase
  • Addition-elimination

Addition-elimination after protonation
23
Mechanisms for the Rearrangement of the
(R)-Methylmalonyl Radical to the Succinyl Radical
(RS coenzyme A)
24
Calculation of Reaction Pathways
  • Ab initio molecular orbital calculations were
    carried out on a model reaction, the degenerate
    rearrangement of the 3-propanal radical

(cf. D. M. Smith,, B. T. Golding, and L. Radom,
J. Am. Chem. Soc., 1999, 121, 1037 and 1383)
25
Possible Mechanisms for the Degenerate
Rearrangement of the 3-Propanal Radical
26
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.
  • Can partial protonation by a
  • weaker acid (H-X) help?

27
Quantifying Partial Protonation
The effect of protonating the 3-propanal radical
by three different acids was investigated using
MO theory
28
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 CO
    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.

29
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.

30
Active Site of Methylmalonyl-CoA Mutase
Substrate
Nearest histidine N - substrate CO separation
2.95 Å Cobalt - substrate CO separation 8.5 Å
Tyr89
His244
Corrin
Arg207
(F Mancia and P R Evans, Structure, 1998, 6,
711)
31
Possible Rationalisations for (a) the Inversion
Pathway of Glutamate Mutase (b) the Retention
Pathway of Methylmalonyl-CoA Mutase
In path b, migration to the Re face may be
blocked by deoxyadenosine.
32
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-recombinatio
    n 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).
33
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
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