How Do Enzymes Perform and Control Radical Chemistry?

Bernard T Golding

Department of ChemistryUniversity 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)

• -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

ENZYME a b X YDiol dehydratase H or Me H, Me, CF3 OH OHEthanolamine ammonia lyase H H or Me NH2 OHMethylmalonyl CoA mutase H H COSCoA CO2HGlutamate mutase H H CH(NH3+)CO2- CO2H2-Methyleneglutarate mutase H H C(=CH2)CO2- CO2H

C C

H

a b

Y H

X

C C

X

a b

Y H

H

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.

SS

SH2N

H

HO2C

H

CO2

H2C

HO2C

H

CO2

NH2

H

pro-S

(review: W Buckel and B T Golding, Chem Soc Revs, 1996, 26, 329-337)

Structure of Coenzyme B12

D C

BA

g

e

d

cb

a

f

R

Co

N

N

Me O

OP

O

O

NH

H2NOC

CONH2

Me

H2NOC

CONH2

CONH2

Me

Me

Me

Me

Me

H

Me

MeN N

NN

HO

CH2OH

-OO

Me

CONH2

Me

Co

HH

Ado

O

HO OH

H2C N

N

NN

NH2

R (5'-deoxyadenosyl) =

adenosylcobalamin AdoCH2-Cbl

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.

pro-S

HH

Ado

Co

H2C

HO2C

H

CO2

NH2

H

H2N

H

HO2C

H

CO2

adenosylcobalamin

SS

S

Reaction Pathway for Glutamate Mutase

HH

Ado

Co Co

AdoH

H

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:

AdoCH2AdoCH3

AdoCH3AdoCH2

CO2-

H

-O2CH

HNH2

-O2C

H

HNH2

CO2-

CH2-O2C

CO2-

H

H2N

CH2-O2C

CO2-

H

H2N

Hpro-S

S

S

S

Possible Rearrangement Mechanisms for Glutamate Mutase

Fragmentation-recombination pathway:

Note that this mechanism has strict stereoelectronic requirements: the -bond undergoing cleavage must be properly aligned with the p-orbital of the 4-glutamyl radical.

-O2C

H

HNH2

CO2-

-O2C

CO2-

H

H2N

H

HCO2

-

H

H2N

-O2C

H

Possible Rearrangement Mechanisms for Glutamate Mutase

CO2-

H

-O2CH

HNH2

-O2C

H

CO2-

N

X

CH2-O2C

CO2-

N

X

CO2-

H

-O2CH

HN

X

CO2-

H

-O2CH

N

X

-O2C

CO2HN

X

CH3-O2C

CO2-

H

H2N

Addition-elimination via an intermediate imine:

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:

-O2C

H

HNH2

CO2-

EPR Study of Glutamate Mutase

EPR spectra of the radical species derived from incubating glutamate mutase and coenzyme B12 with 13C-labelled (S)-glutamate.

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)

2-Methyleneglutarate Mutase

• 2-Methyleneglutarate mutase from Clostridium barkeri catalyses the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate:

K = 0.06

pro-RMe

-O2CH CO2

--O2C

H

-O2CH

2 AdoCH2-Cbl

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

R

R

.. .

-O2CH

HH

CO2-

-O2C

H

-O2C

HH

-O2C

H

-O2C

-O2CH

Me

CO2-

-O2CH

H

-O2C

1a 2a

3 5 4

Equilibration of 2-methyleneglutarate 1a and (R)-3-methylitaconate 2a and their corresponding radicals 3 and 4 via cyclopropylcarbinyl radical 5:

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.

-O2CH

Me

CO2-

D

H

-O2C

H

-O2C

H

D.

-O2CH

Me

CO2-

D

H

?? via

2b 2cZ E

1

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 (‘fragmentation-recombination’) in which the substrate-derived radical 3 fragments to acrylate and the 2-acrylate radical 6 (path b).

6

453

2a1a-O2CH

H

-O2C-O2C

H

Me

CO2-

-O2C

H

-O2C

-O2C

H

-O2C

HH

-O2CH

HH

CO2-

...

R

-O2C H

HH

.-O2C

path a

path b

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.

• For the fragmentation pathway, it suffices to achieve maximal overlap between the p orbital at C-4 and the critical C-2/C-3 -bond.

• The two alternative mechanisms can in principle be distinguished by the conformation of the substrate bound to the enzyme.

43

2

Position of thisgroup can differin the two pathwaysCritical bond

-O2C

H

-O2C

D -O2C

H

-O2C

H D

R

-O2C H

HH

-O2C

D

path a

path b

Methylmalonyl-CoA Mutase

This human enzyme converts the (R)-isomer of methylmalonyl-CoA to succinyl-CoA (RS = coenzyme A):

RCH2

O

RS

HO2C

H

O

SR

H

HO2CHpro-R

from propionate, atoxic product of the degradation of fats

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:

R

H

O2CH

O

SRpro-R

CH2O2C

H

HO

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:

C

O

SCoA

CO2- CO2

-

HO

SCoA

H2C

C

CO2-

H

O

SCoA

Radical corresponding to methylmalonyl-CoA

Radical corresponding to succinyl-CoA

Pathways for Methylmalonyl-CoA Mutase

Addition-elimination:

Addition-elimination after protonation:

.CO2

-

HO

SCoA.

CO2-

H

SCoAO

.H2C

C

CO2-

H

O

SCoA

.CO2

-

HHO

SCoA

.CO2

-H

SCoAHO

.H2C

C

CO2-

H

HO

SCoA

Mechanisms for the Rearrangement of the (R)-Methylmalonyl Radical to the Succinyl Radical

Re face

- H+ H

TSTS

TS

TS

.

..

..

.

.

..

O

SR

HO2C

HO

SRHO2C

HO2C

RS OHOH

RS

HO2C

O

RS

HO2C

O

RS

HO2C

O

SRHO2C

HHO2C

RS OO

RS

HO2C

(RS = coenzyme A)

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)

Pathway H# (kJ mol-1)

Fragmentation-recombination

96

Addition-elimination 47

Addition-elimination afterprotonation

10

.CH2

C

HH

O

H

H2C

C

HH

O

H. intermediate radical

Possible Mechanisms for the Degenerate Rearrangement of the 3-Propanal Radical

O O O

O

O

OH OH HO

O

. .

.

.

. .

. ..

TS

TS

TS TS

+ H - H

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?H2C

C

CO2-

H

O

SCoA

H X

Quantifying Partial Protonation

Acid Proton affinity

of conjugate

base (kJ mol-1)

C=O……HX

distance (Å)

C=O distance

(Å)

Rearrangement

energy barrier

(kJ mol-1)

None 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

Full protonation 0.976 1.299 10.0

The effect of protonating the 3-propanal radical by three different acids was investigated using MO theory:

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

(F Mancia and P R Evans, Structure, 1998, 6, 711)

His244

Corrin

Tyr89

Substrate

Arg207

Nearest histidine N - substrate C=O separation = 2.95 Å

Cobalt - substrate C=O separation = 8.5 Å

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.

b

a

S

S

S

pro-S

CH2-O2C

CO2-

H

H2N

H

CH2-O2C

CO2-

H

H2N

-O2C

H

HNH2

CO2-

CO2-

H

-O2CH

HNH2

AdoCH2

. AdoCH3

. .

AdoCH3.

AdoCH2

Re face

R

Re faceAdoCH2.AdoCH3

CH2

O

RS

-O2C

H

CH2

O

RS

-O2C

O

SR-O2C

H

..O

SR

H

-O2CHpro-R

AdoCH3.AdoCH2

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