14.8. Bioinorganic Catalysis CobaTamin Reactions Cobalamin-Catalyzed Enzymatic Reactions

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1 Cobalamin-catalyzed reactions are generally classified into two groups: methylcobalamin-dependent reactions (Table 1, entry 1 to 3) and coenzyme B i2 -dependent rearrangements (Table 1, entry 4 to 11). The first group includes the biosynthesis of methionine from homocysteine, the reduction of CO 2 to acetic acid via an acetyl-coa pathway, and the biosynthesis of CH 4 also via an acetyi-coa pathway 1. In the synthesis of methionine, cob(i)alamin is methylated by methyl tetrahydrofolate [MeH 4 THF 2 (equation (a)], followed by the transfer of CH 3 + from methylcobalamin to homocysteine 1,3. The biosynthesis of acetate was believed to form a similar methyl corrinoid which transfer CH 3 to CO 2 (carboxylation of methylcobalamin) 4. However a reduction pathway - involving methylcorrinoid and CO via formation of an acetyl radical (carbonylation) has recently been proposed 5. In this process, one molecule of CO 2 is reduced to the methyl level and appears as MeH 4 THF. Again methylcobalamin, formed by MeH 4 THF and B 12s, reacts with another CO (derived from CO 2 ), via radical intermediates, followed by recombination of an acetyl radical and B 12r to give acetylcobalamin 6-9. However, release of an acetyl group, via reaction of acetylcobalamin with a thiol to give a thioester, analogous to acetyl-coa, is still not demonstrated. The role of cobalamin in the biosynthesis of CH 4 is more complex. Cobalamin serves as an electron donor for enzymatic reduction of methyl-com to CH 8 4. Dithiothreitol or SnCl 2 reduces B 12a to B 12r, which is known to disproportionate subsequently to B i2a and B 12s. The latter then acts as a stronger reducing agent in the reduction. Inhibition of CH 4 production by propyl iodide is attr buted to formation of Pr Co species, which suppresses electron transfer 8. Attempts have been made to mimic the above reactions under nonenzymatic conditions. Methylcobalamin methylates homocysteine, but the reaction is a free radical process 10. Also, MeH 4 THF does not methylate cobaloxime(i) or B i2s. N-Methylamines are also unreactive toward Co(I) species'. Tetraalkylammonium compounds alkylate Co(I) nucleophiles to form alkylcobalt compounds 11 ; it is possible that MeH 4 THF is protonated by the enzyme and this methylates the cobalamin. Methylation in low yield of cobalamin(i) by MeH 4 THF at low ph 12 has been disputed 1. The second group of enzymatic reactions involves coenzyme B 12. All but one (Table 1, entry 11) can be described as in equation (b). Spectroscopic studies indicate homolytic cleavage of the enzyme bound coenzyme to a deoxyadenosyl radical (ACH 2 ') and B 12r as a common characteristic of these enzymatic reactions'. Isomerization, described above, involves an apparent intramolecular 1,2-shift of a hydrogen and an electronegative group (X = OH, NH 2, Table 1, entry 4 to 6) or a carbon skeleton (Table 1, entry 7 to 10). However, in the nucleotide reductase system, coenzyme B 12 has a unique role of radical initiator in a radical chain mechanism rather

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3 than acting as an intermediary hydrogen transfer agent 13,14. Unlike the other 1,2-shift reaction, hydrogen derives from an exogenous thiol reductant. 15 The migrating atom of the substrate is equil brated with the two hydrogens of the 5'-carbon atom of deoxyadenosine (ACH 2 -) bound to cobalt (Scheme 1). In the reaction in eq. b, ESR experiments show Co(II) as an intermediate in the these transformation 1. B 12r existence has also been confirmed by X-ray crystallography in the solid state 16 and EXAFS spectroscopy in solution 17. B 12r contains 5-coordinate low-spin Co(II). Structurally, the Co(II) corrin is strikingly similar to that of the coenzyme B 12 16,17. The axial base of the coenzyme was thought previously to play a key role in Co C bond weakening by pushing the corrin ring upward. The bond dissociation energy (BDE) in coenzyme B 12 is only 23 kj/mol lower than in adcnosylcobinamide (base-off form) 18. It is very likely that binding the coenzyme to the enzyme through the benzimidazole base increases the protein interactions both sterically and electronically, so that the bond is readily cleaved to give ACH 2. 18,19. Tlie carbon skeleton rearrangements [Table 1, entry 7 to 10; equation (b), X = COSCoA, CHN H 3 CO 2, C(=CH2 )C0 2, CH2 COSCOA] are revers ble. The isobutyl CoA mutase reaction was discovered recently; little is yet known about this reaction 20. Many model systems have been developed for the methylmalonyl-coa mutase re action. With simple models, low yields of rearranged product are obtained 21,22. When the thioester 1 reacts with B 12s, up to 70% of the rearranged product 2 forms 23,24. A free radical pathway involving 3 and 4 was proposed 24. The key intermediate 3 was generate! later by reaction of 1 with n-bu 3 SnH, in the absence of coenzyme, and 4 was trapped by H.. Therefore 2 is the only product obtained 25,26. Spontaneous 1,2-migration of the thioester group was first demonstrated in the model free radical 26. Rearrangement in methylmalony-coa mutase reaction may occur at the free radical stage. Although cobalt participation in the rearrangement cannot definitely be excluded, it seems unlikely 25. The principal role of coenzyme B 12 in the rearrangement process appears to be as a free radical precursor, a role that depends on the weakness of the cobalt-carbon bond of coenzyme B Both free organic radical and organocobalt pathways have been postulated (Scheme 2) in the rearrangement of 2-methyleneglutarate to methylitaconate. 1,2-Migration of an acrylate group in a free radical intermediate is precedented via a cyclopropyl methyl

4 CobaIamin Reactions radical intermediate 27,28. Although cobalt participation cannot be excluded, the free radical mechanism appears more favorable. However, some model studies suggest a cobaltparticipation pathway (Scheme 3). 1-Methylbut-3-enylcobaloxime (5) rearranges to 2- methy but-3-encyclobaloxime (6) 29-30, via 3-methylcyclopropylcarbinylcobaloxime, giving a 1:10 equilibrium mixture of compounds 5 and 6. Similarly, the suggested intermediate 3-methylcyclopropylcarbinylcobaloxime rearranges to 5 and 6 to give the same equilibrium mixture 31. Rearrangement of glutamic to methylaspartic acid is one of the four rearrangement reactions (Table 1, 7 to 10) catalyzed by coenzyme B 12, which involves carbon skeleton rearrangements. Furthermore, the migrating group is a saturated carbon. Thermolysis of model compound 7 produces 8 and 9; no rearranged glutamic acid was detected 32.

5 Cobalamin Reactions Experiments designed to mimic vitamin B 12 holoenzyme by introducing some hydrophobic cavity have been initially successful toward elucidating this rearrangement mechanism. Hydrophobic cage effects and molecular aggregates favor formation of rearranged products, because in both cases the thermolysis-generated free radicals are stabilized Many attempts made to induce rearrangement of 10 (Scheme 4) with B, 2s were unsuccessful. However, under free radical conditions (initiated by n-bu 2 SnH) rearrangement occurs (Scheme 4) 32. The above results suggest a free radical mechanism for the enzymatic reactions. The dioldehydrase reaction involves formation of aldehyde from 1,2-diols (glycols). The ethanolamine ammonia lyase and aminomutase reactions are similar. The favored mechanism is shown in Scheme 5. Like other coenzyme catalyzed rearrangements, hom-. olytic cleavage of the Co C bond of coenzyme B 12 produces B 12r and ACH 2 and isotopic scrambling of hydrogen at the C5' carbon of the deoxyadenosyl group confirms the involvement of this radical in hydrogen abstraction.

6 14.8. Bioinoraanic Catalysis Recent studies have progressed toward understanding the mechanism of the rearrangement step. In a model system (11 in Scheme 6), the vicinal diol group, protected as its carbonate, is activated by MeO - in MeOH; rearrangement reaction yielded 100% Co(Il) and 95% CH 3 CHO (Scheme 6) 36. The added axial base 1,5,6-trimethylbenzimidazole drastically alters the observed products (Scheme 6). No CH 3 CHO or Co(II)CH 2 CHO, the supposed cobalt participation intermediate, was observed 36. Recently a carbonate-protected form of a B 12 -Dound 1,2-diol 12 was prepared and activated as above. Only B 12s and CH 3 OCO 2 CH 3 were detected. As expected from the model system (Scheme 7), no CH 3 CHO, B 12r or formylcobalamin (key intermediate for cobalt participation) were detected 37. These products are also obtained in a separate experiment using pulse radiolysis generated HOCH 2 C. H(OH) radicals in the presence of B 12r 38,39. The currently available enzymatic and chemical model supporting evidence strongly argues for a pathway involving nonparticipation of the cobalt. Cobalt participation can lead to a redox side reaction 37. Rearrangement of an a-hydroxy radical occurs 36,40,41.

7 14.8. Bioinoraanic Catalysis Computational studies 41 of a-oh radicals indicate that protonation of the hydroxy group lowers significantly the barrier to OH migration (Scheme 8), and suggests a possible acidic protein binding site in the enzyme 40. Finally, a yet unproved radical chain mechanism has been proposed 40. (L. Y. XIE, P. F. ROUSSI, D. H. DOLPHIN) 1. B. T. Golding, in Comprehensive Organic Chemistry, Vol. 5, D. H. R. Barton, W. 0. Ollis, eds., Pergamon Press, Oxford, 1979, p Where H 4 THF = tetrahydrofolic acid MeH 4 THF = N 5 -Methyltetrahydrofolic acid. 3. R. T. Taylor, in B 12, Vol. 2, D. Dolphin, ed., Wiley-Interscience, New York, 1982, p L. G. Ljungdahl, H. G. Wood, in B 12, Vol. 2, D. Dolphin, ed., Wiley-Interscience, New York, 1982, p B. Krautler, Helv. Chim. Acta, 67, 1053 (1984). 6. H. G. Wood, S. W. Ragadale, E. Pezacka, Trends Biochem. Sci., 11, 14 (1986). 7. J. G. Zeikus, R. Kerby, J. A. Krzycki, Science, 227, 1167 (1985). 8. D. Ankel-Fuchs, R. K. Thauer, Eur. J. Biochem., 156, 171 (1986). 9. G. Fuchs, FEMS Microbio. Lett., 39, 181 (1986). 10. D. Dodd, M. D. Johnson, J. Organomet. Chem., 52, 58 (1973). 11. G. Costa, A. Puxedu, E. Reisenhofer, J. Chem. Soc., Dalton Trans., 2034 (1973). 12. H. Rudiger, Eur. J. Biochem., 21, 264 (1971). 13. G. W. Ashley, G. Harris, J. Stubbe, J. Biol. Chem., 261, 3958 (1986). 14. J. Stubbe, Mol. Cell Biochem., 50, 25 (1983). 15. W. S. Beck, Am. J. Hematol., 34, 83 (1990). 16. B. Krautler, W. Keller, C. Kratkey, J. Am. Chem. Soc., Ill, 8936 (1989). 17. I. Sagi, M. D. Wirt, E. Chen, S. Frisbie, M. R. Chance, J. Am. Chem. Soc., 112, 8639 (1990). 18. B. P. Hay, R. G. Finke. J. Am. Chem. Soc., 109, 8012 (1987). 19. B. T. Golding, in B 12, Vol. 1, D. Dolphin, ed., Wiley-Interscience, New York, 1982, p D. Gani, D. O'Hagan, K. Reynolds, J. A. Robinson. J. Chem. Soc., Chem. Commun., 1002 (1985) K. Reynolds, J. A. Robinson,/. Chem. Soc., Chem. Commun., 1831 (1985) K. Rey-

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