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Enzymatic Radical Chemistry: Mechanisms and Control

This study explores the performance and control of radical chemistry in enzymatic reactions, focusing on examples such as coenzyme B12-dependent reactions and glutamate mutase. Through various tools and techniques, the mechanisms of these reactions are elucidated.

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Enzymatic Radical Chemistry: Mechanisms and Control

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  1. How Do Enzymes Perform and Control Radical Chemistry?Bernard T GoldingDepartment of ChemistryUniversity of Newcastle upon TyneNewcastle 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 man’s 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)-glutamate. 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 4via 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 3fragments 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 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.

  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 C=O separation = 2.95 Å Cobalt - substrate C=O 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-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).

  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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