Glutamate mutase catalyzed reactions

Theoretical studies that has investigated the homolysis step in different enzymatic systems [68-70] reveal that small models comprising only the corrin ring and two ligands are insufficient and that inclusion of more amino acids are essential to stabilize the radical intermediates. Recently, a QM/MM study of the initial phase of the glutamate mutase-catalyzed reaction found a large electrostatic stabilization by the surrounding protein [70], In our study of MCM we employed the ONIOM QM MM approach to reveal the role of the protein in the rupture of the Co—C5 bond [29],... 

In both cases the reaction proceeds with retention of configuration at C-2 and with stereochemical specificity408 for one of the two hydrogens at C-l. The reaction catalyzed by methylmalonyl-CoA mutase likewise proceeds with retention of configuration at C-2 (Table 16-1)409 but the glutamate mutase reaction is accompanied by inversion (Eq. 16-28). 

FIGURE 16. Concerted and kinetically coupled mechanisms for CooC bond homolysis and hydrogen atom abstraction, illustrated for the reaction catalyzed by glutamate mutase. Either mechanism could give rise to the deuterium isotope effects observed in pre-steady state stopped flow experiments. 

The reaction catalyzed by glutamate mutase stands out as the only case in which the migrating carbon is sp hybridized, and hence rearrangement through a cyclic intermediate is not possible (pyridoxal phosphate is not a cofactor). A plausible mechanism for the rearrangement of glutamyl radical... 

FIGURE 24. Free energy profile for the reaction catalyzed by glutamate mutase. E, enzyme M, methylaspartate, Gly, glycine A, acrylate G, glutamate. 

Chih, H.-W., and Marsh, E. N. G., 1999, Pre-steady state investigation of intermediates in the reaction catalyzed by adenosylcobalamin-dependent glutamate mutase. Biochemistry 38 in press. 

Glutamate mutase was discovered by Barker, who showed that the enzyme catalyzes the equilibration of (i j-glutamate with (25,36 )-3-methylaspaitate (Entry 1, Table 1 AG° = + 6.3 kJ mol K = 0.095) (for a review of glutamate mutase see Reference 7). This reaction is one of three distinct methods that nature uses to ferment glutamate to butyrate (5). Surprisingly, they all proceed through intermediate radicals. The coenzyme B 12-dependent fermentation is the only one of the three that... 

Cheng MC, Marsh ENG. Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase. Biochemistry 2007 46 883-889. 

Enzymes catalyzing carbon skeleton rearrangements were the first to be recognized as coenzyme B 12-dependent. The first was glutamate mutase (GM), discovered in 1960, and the nature of the skeletal rearrangement in the reaction of GM inspired the identification of coenzyme B12 as the coenzyme for the apparently similar reaction of... 

Table I is a summary of all of the reactions known to date which require vitamin B12 coenzyme (i). The reaction catalyzed by glutamate mutase, the first reaction in which vitamin B12 was known to be involved, was discovered by Barker and his coworkers. The second reaction is the only one of these which also occurs in mammals all other reactions occur in bacteria. Much of the experimental data described here will be derived from the enzyme called dioldehydrase. An additional reaction which is not shown is the conversion of nucleotides to deoxynucleotides. The conversion of —CHOH— to — CH2— is in some way very similar to the reaction catalyzed by dioldehydrase. Vitamin B12 coenzyme-depend-...

Fig. 18 Glutamate mutase (GM) interconverts (S)-glutamate and (2S,3S)-3-methylaspartate. Proposed reaction mechanism of the carhon skeleton rearrangement, catalyzed by GM involving H-atom abstraction (step a), radical rearrangement (step b) and back transfer of H-atom (step c). (The experimentally supported substrate triggered formation of the 5 -deoxy-5 -adenos)d radical and of cob(ll)alamin (23, Bi2r) by homolysis of protein bound AdoCbl (2) is omitted here, see Fig. 16 [173,175,176])...

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]. 

Much has been done, said, and written in enzyme kinetics and I will mention only a few things. The enzymes are usually selective they catalyze only a single reaction or only one type of reaction. While enzymes generally speed up the reactions, in comparison to the same reaction conducted in the laboratory, the enzymes that are not very selective are usually relatively slow. On the other hand, certain highly selective enzymes, like carbonic anhydrase or glutamate mutase, can speed up the reaction conducted under laboratory conditions by a factor of 10 -lO, that is, trillion-to quadrillion-fold. No man-made catalyst matches this efficiency. Thousands of enzymes are known today they are catalogued into six major categories, in relation to the type of chemical reaction they catalyze. Each enzyme is identified by its enzyme code number, or E.C. number [5]. 

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. 

Synthesis of glutamate removes a-ketoglutarate from the TCA cycle, thereby decreasing the regeneration of oxaloacetate in the TCA cycle. Because oxaloacetate is necessary for the oxidation of acetyl CoA, oxaloacetate must be replaced by anapierotic reactions. There are two major types of anapierotic reactions (1) pyruvate carboxylase and (2) the degradative pathway of the branched-chain amino acids, valine and isoleucine, which contribute succinyl CoA to the TCA cycle. This pathway uses B12 (but not folate) in the reaction catalyzed by methylmalonyl CoA mutase. 

The transamination of P-aminoisobutyrate to form methylmalonate semialdehyde requires pyridoxal phosphate as a cofactor. This reaction is similar to the conversion of ornithine to glutamate y-semialdehyde. Then NAD+ serves as an electron acceptor for the oxidation of methylmalonate semialdehyde to methylmalonate. The conversion of methylmalonate to methylmalonyl CoA requires coenzyme A. The final reaction, in which methylmalonyl CoA is converted to succinyl CoA, is catalyzed by methylmalonyl CoA mutase, an enzyme that contains a derivative of vitamin B12 as its coenzyme. 

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