Coenzyme radical formation

FIGURE 16 (Top) A family of rearrangement reactions that depend upon free radical formation involving an enzyme-bound form of the vitamin B12 coenzyme S -deoxyadenosylcobalamin (Fig. 7). The rearrangement of (R) methylmalonyl-CoA to succinyl-CoA (the opposite of the reaction shown here) is one of the two essential vitamin Bi2-dependent reactions in the human body, and plays an important role in fatty acid oxidation, as is indicated in Fig. 12. 

Flavin adenine dinucleotide. See FAD Flavin adenine diphosphate. See FAD Flavin coenzymes 766,780 - 795 modified 788, 789 reduced 794 Flavin radicals 792 color of 794 formation constant 794 Flavocytochrome b2 782, 794, 847 Flavodoxins 793, 799, 800 Flavoprotein(s) 513, 788... 

The best-studied enzymes to date that contain glycyl radicals are pyruvate formate-lyase (PFL) and a ribonucleoside triphosphate reductase (ARR), both isolated from anaerobically growing E. coli. These enzymes play central roles in the anaerobic metabolism of the bacterium. The first catalyzes the reversible formation of acetyl-CoA and formate from pyruvate and coenzyme A, while the second is responsible for synthesizing the deoxyribonucleotide monomers of the polymer DNA. It is intriguing to note that formate, a product of the PFL reaction, is a substrate for the ARR. It supplies the reducing equivalents needed for each round of deoxynucleotide synthesis. ... 

In mammals, there are only three vitamin B12 -dependent enzymes methionine synthetase, methylmalonyl CoA mutase, and leucine aminomutase. The enzymes use different coenzymes methionine synthetase uses methylcobal-amin, and cobalt undergoes oxidation during the reaction methylmalonyl CoA mutase and leucine aminomutase use adenosylcobalamin and catalyze the formation of a 5 -deoxyadenosyl radical as the catalytic intermediate. 

The role of coenzyme B12 as cofactor of enzymatic rearrangement reactions requires the (reversible) formation of the 5 -adenosyl radical by homolysis of the organometal-lic bond of (3) (estimated bond-dissociation energy ca. 30kcalmol to be accelerated there by a factor of... 

ESR studies 112,114-118a) are also consistent with the formation of free radicals upon photolysis of alkylcobalamin and coenzyme Bi2- For example, Lappert and co-workers 116) demonstrated that homolysis of the cobalt-alkyl bond occurs upon photolysis of coenzyme B12 and ethylcobalamin by trapping the S -deoxyadenosyl and ethyl radicals produced with (CH3)3CN0. They were able to detect the spin-trapped (CH3)3CN(0)R radicals by ESR spectroscopy. Homolysis of the cobalt-methyl bond was also shown to occur upon anaerobic photolysis of methylcobalamin 117). However, the presence of traces of oxygen in the methanol solvent was shown to affect signiflcantly the photochemistry of methylcobalamin 118). Indeed, under those conditions, the 5,S-dimethyl-1-pyrroline iV-oxide (DMPO) spin adducts of both the methyl and hydrogen radicals, 113 and 114, respectively, were detected by ESR spectro-... 

This enzyme s role in humans is to assist the detoxification of propionate derived from the degradation of the amino acids methionine, threonine, valine, and isoleucine. Propionyl-CoA is carboxylated to (5 )-methylmalonyl-CoA, which is epimerized to the (i )-isomer. Coenzyme Bi2-dependent methylmalonyl-CoA mutase isomerizes the latter to succinyl-CoA (Fig. 2), which enters the Krebs cycle. Methylmalonyl-CoA mutase was the first coenzyme B -dependent enzyme to be characterized crystallographically (by Philip Evans and Peter Leadlay). A mechanism for the catalytic reaction based on ab initio molecular orbital calculations invoked a partial protonation of the oxygen atom of the substrate thioester carbonyl group that facilitated formation of an oxycyclopropyl intermediate, which connects the substrate-derived and product-related radicals (14). The partial protonation was supposed to be provided by the hydrogen bonding of this carbonyl to His 244, which was inferred from the crystal structure of the protein. The ability of the substrate and product radicals to interconvert even in the absence of the enzyme was demonstrated by model studies (15). 

A second, and more chemical, verification is due to Finke et al.,21 who also invented the descriptive phrase persistent radical effect and gave a prototype example to the extreme. The thermal reversible 1,3-benzyl migration in a coenzyme B12 model complex leads to the equilibrium of Scheme 9. Earlier work had shown that the reaction involves freely diffusing benzyl and persistent cobalt macrocycle radicals, but the expected self-termination product bibenzyl of benzyl was missing. Extending the detection limits, the authors found traces of bibenzyl and deduced a selectivity for the formation of the cross-products to the self-termination products of 100 000 1 or 99.999%. Kinetic modeling further showed that over a time of 1000 years only 0.18% of bibenzyl would be formed, and this stresses the long-time duration of the phenomenon. 

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