Coenzyme Bi2-dependent enzymes

About ten coenzyme Bi2-dependent enzymes are now known (see Table 1) [6, 25,153,173]. These enzymes are four carbon skeleton mutases (methyl-malonyl-CoA mutase [174], glutamate mutase [175,176], methylene glutarate mutase [175] and isobutyryl-CoA mutase [177]), diol dehydratase [178], 

To conclude the coenzyme B -dependent enzymes all appear to based on the reactivity of bound organic radicals, which are formed (directly or indirectly) by a H-atom abstraction by the S -deoxy-S -adenosyl radical, that originates form the homolysis of the Co-C bound of AdoCbl (2). In these enzymatic reactions, the 5 -deoxy-5 -adenosyl radical is the established reactive partner in the actual enzymatic reaction, so that 2 should be looked at as a pre-catalyst (or catalyst precursor) [75]. Coenzyme B12 (2) might 

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The known coenzyme Bi2-dependent enzymes all perform chemical transformations in enzymatic radical reactions that are difficult to achieve by typical organic reactions. Homolytic cleavage of the Co bond of the protein-bound coenzyme B12 (3) to a 5 -deoxy-5 -adenosyl radical (9) and cob(n)alamin (5) is the entry to reversible H-abstraction reactions involving the 5 -position of the radical (9). Indeed, homolysis of the Co bond is the thermally most easily achieved transformation of coenzyme B12 (3) in neutral aqueous solution (with a homolytic (Co-C)-BDE of about 30 kcal mol ). However, to be relevant for the observed rates of catalysis by the coenzyme B12-dependent enzymes, the homolysis of the Co-C bond of the protein-bound coenzyme (3) needs to be accelerated by a factor of about 10 , in the presence of a substrate. Coenzyme B12 might then be considered, first of aU, to be a structurally sophisticated, reversible source for an alkyl radical, whose Co bond is labihzed in the protein-bound state (Figure 8), and the first major task of the... 

Bnckel W, Golding BT. Glntamate and 2-methyleneglntarate mntase from microbial cnriosities to paradigms for coenzyme Bi2-dependent enzymes. Chem. Soc. Rev. 1996 26 329-337. 

N., Banerjee, R., Spiro, T. G. (1998) Resonance Raman spectra of methylmalonyl-Coenzyme A mutase, a coenzyme Bi2-dependent enzyme, reveal dramatic change in corrin ring conformation but little change in Co-C bond force constant in the cofactor upon its binding to the enzyme, J. Am. Chem. Soc. 120, 9947-9948. 

As in the reactions of all coenzyme Bi2-dependent enzymes, the observable free radicals display spin-spin coupling with low-spin Co in cob(II)alamin and are quantitatively characterized as diradicals or radical triplet species. As in DDH, this coupling is weak in EAL, and quantitative analysis indicates a separation of... 

Most coenzyme Bi2-dependent enzymes lose activity during prolonged reactions with substrates. 5,6-LAM is especially susceptible to substrate-dependent suicide inactivation. Based on extensive biochemical analysis, the chemical course of this process involves proton-coupled electron transfer leading to quenching of the putative substrate radical intermediates. Lysine-dependent suicide inactivation displays the following properties ... 

Like glutamate, 2-methyleneglutarate can be fermented in Clostridia, albeit by a different pathway,and the reaction of MGM is the first step. Less information about MGM is available than other coenzyme Bi2-dependent enzymes. The stereochemistry, purification, and molecular characterization are available. A substrate-based free radical has been observed by EPR spectroscopy. The reaction mechanism is not known, although it likely follows the generic pathway of hydrogen abstraction by the 5 -deoxyadenosyl radical to form a substrate-derived radical, which rearranges to a product-related radical that is quenched by hydrogen transfer from 5 -deoxyadenosine. The mechanism of radical isomerization remains to be proven. ... 

The observation of a kinetically competent cysteinyl-thiyl free radical intermediate implicated Cys408 in a critical mechanistic role. As in other coenzyme Bi2-dependent enzymes, the unpaired electron of the radical was spin coupled to Co of cob(II)alamin. The structure of the thiyl radical was rigorously proven by isotope-edited EPR analysis, and spectral simulations showed that the thiyl radical resided at 5—7 A from Co of cob(II)alamin. The emergence of the radical triplet signal required adenosylcobalamin and dGTP in addition to RTPR. 

Class II RTPR has in common with other coenzyme Bi2-dependent enzymes the use of the 5 -deoxyade-nosyl radical from adenosylcobalamin as a radical initiator. The elimination of water from the 3 -radical intermediate might in part be analogous to the elimination of water in the reaction of DDH. The variant function of RTPR can be viewed as foreshadowing the wider functions of the 5 -deoxyadenosyl radical in other radical reactions, in particular the radical SAM enzymes. ... 

The demonstrated ability of vitamin Bi2s to form 7r-complexes with electron-accepting olefinic ligands shows that it may be difficult to detect the presence of the Co(I) nucleophile in coenzyme Bi2-dependent enzymes. Apart from the fact that the stationary concentrations of vitamin Bi2s are expected to be altogether too low for direct observation, the spectroscopic properties of enzyme-bound vitamin Bi2s need not be necessarily identical with those of the free compound. 

Coenzyme Bi2-dependent enzymes Co Homolytic Co-C bond cleavage to generate Co" plus radical... 

The homolytic cleavage of the Co - C bond of the protein-boimd organo-metallic cofactor AdoCbl (2) is the initial step of the coenzyme Bi2-catalyzed enzymatic reactions. Halpern quoted that adenosyl cobamides can be considered as reversibly functioning sources for organic radicals [119]. A neutral aqueous solution of 2 is remarkably stable with a half-Ufe of 10 s (in the dark at room temperature), but decomposes, mainly with the homolysis of the Co-C bond, at higher temperatures [119,123]. The coenzyme B12-catalyzed enzyme reactions occur with maximal rates of approximately 100 s [173,239]. Rapid formation of Co(ll)corrins occurs only with addition of substrate to a solution of holoenzyme (or of apoenzymes and 2), as demonstrated in most of the known coenzyme Bi2-dependent enzymes, e.g., in methyl-malonyl-CoA mutase [121], glutamate mutase [202] and ribonucleotide reductase [239]. 

Thus, an intriguing feature of the coenzyme Bi2-dependent enzymes is the dramatic (> lO -fold) labihzation of the bound organometallic cofactor towards homolysis of the Co-C bond [119,123,173]. The mechanism of the enzyme (and substrate-induced) labihzation of this Co-C bond is stiU a key problem, and much discussed, in Bn-chemistry. Evidence for covalent restructuring of the bound cofactor (except for the formation of the base-off/His-on form in the carbon skeleton mutases) is not available [75,119,123, 173,194]. In addition protein and solvent molecules can only weakly stabihze a radical center [240]. Steric distortions of the protein-bound cofactor were discussed as means for the enhanced rate of Co - C bond homolysis [51,119, 163,217]. Halpem s theory of an upwards conformational distortion of the... 

Heptamethylcob3rrinate cobester (13) and peripherically functionalized derivatives of 13 were used as catalysts to mimic the rearrangement catalyzed by the coenzyme Bi2-dependent enzyme methylmalonyl-CoA mutase (see Fig. 13) (45). In these studies, the reductive transformation of bromomethyl-malonates to succinates, catalyzed by 13, and similar rearrangement reactions were observed (45). 

Two views of the structure of the coenzyme Bi2-dependent enzyme glutamate mutase from Gostridium cochlearium, demonstrating binding with methylcobalamin (center of each molecule). 

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