2-Methyleneglutarate mutase

The first B 12-dependent enzyme to be considered is 2-methyleneglutarate mutase which catalyzes the interconversion of 2-methyleneglutarate with (/ )-3-methylitaconate [67]  

This transformation is part of a microbial metabolic pathway in which nicotinate is broken down into ammonia, CO2, acetate and pyruvate [16, 68]. Accepting the bound free-radical hypothesis implies that the cmcial radical rearrangement step can be represented by reaction 4, in which a 2-methyleneglutarate-derived radical (1) is transformed into an (/ )-3-methylitaconate-related radical (2) [6]  

There have been two major suggestions for the rearrangement in reaction 4  

Applying the suggested simplifications to the 2-methyleneglutarate-mutase-catalyzed rearrangement results in the system shown in reaction 5  

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KungH-F, LTsai(1971)Nicotinicacidmetabolism. VII.Mechanismsofactionofclostridiala-methyleneglutarate mutase (Bjj-dependent) and methylitaconate isomerase. J Biol Chem 246 6436-6443. 

The structure of the E. coli enzyme (Fig. 16-24) shows methylcobalamin bound in a base-off conformation, with histidine 759 of the protein replacing dimethylbenzimidazole in the distal coordination position on the cobalt. This histidine is part of a sequence Asp-X-His-X-X-Gly that is found not only in methionine synthase but also in methylmalonyl-CoA mutase, glutamate mutase, and 2-methyleneglutarate mutase. However, diol dehydratase lacks this sequence and binds adenosylcobalamin with the dimethylbenz-imidazole-cobalt bond intact.417... 

FIGURE 21. Mechanisms for the rearrangement of substrate radicals in the reactions catalyzed by carbon skeleton mutases. For 2-methyleneglutarate mutase and the acyl-CoA mutases both associative (upper pathway) and dissociative (lower pathway) mechanisms have been proposed (Halpem, 1985 Bucket Golding, 1996), whereas for glutamate only a dissociative mechanism appears feasible. 

Chemaly, S. M., 1994, a-Methyleneglutarate mutase an adenosylcobalamin-dependent enzyme, S. Afr. J. Chem. 47 37947. 

Michel, C., Albracht, S. P., and Buckel, W., 1992, Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri, Eur. J. Biochem. 205 767n773. 

Scheme 5 Two pathways for 2-methyleneglutarate mutase (substrate 2-methyleneglutarate product (R)-3-methylitaconate). Path a Addition-elimination via substrate radical and product radical P with the participation of an intermediate radical I ...

We chose this reaction since it has been proposed as a model for the rearrangement of 2-methyleneglutarate to 3-methylitaconate, catalyzed by the coenzyme-B,2-dependent enzyme, 2-methyleneglutarate mutase [16, 26, 57]. More specifically, equation 2 represents the second step in the addition-elimination pathway (reaction c. Scheme 4) for a 1,2-shift. Additionally, this reaction has been widely studied experimentally [58] and has been described as the most precisely calibrated radical reaction [59]. 

Scheme 6. Two possible pathways for the radical rearrangement catalyzed by 2-methyleneglutarate mutase.

The first is the fragmentation-recombination pathway (see mechanism a. Scheme 4), with acrylate and an acrylate-derived radical as the intermediate state (Scheme 6). This possibility has been suggested only recently, and is based on the reported inhibition of 2-methyleneglutarate mutase by acrylate [47], The second suggested mechanism is the addition-elimination pathway (see reaction c. Scheme 4), with a substituted cyclopropylcarbinyl radical as the intermediate (Scheme 6) [6, 26],... 

As with the 2-methyleneglutarate mutase system, the detailed computational investigation of the methylmalonyl-CoA mutase system is somewhat complex. We therefore continue to use the model system approach, and replace the SCoA and carboxylate groups by hydrogen atoms. This simplification results in the degenerate rearrangement of the 3-propanal radical (8) [69, 76] ... 

Once again a number of different pathways for the degenerate rearrangement of the aminopropyl radical can be considered (Scheme 9) [35], including pathways that are analogous to those examined as models for the reactions catalyzed by methyleneglutarate mutase (Scheme 7) and methylmalonyl-CoA mutase (Scheme 8). 

Kung, H.-F. and T.C. Stadtman. 1971. Nicotinic acid metabolism VI. Purification and properties of alpha-methyleneglutarate mutase (B12-dependent) and methylita-conate isomerase.. Biol. Chem. 246 3378-3388. 

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