Enzymes glutamate mutase

Reitzer, R., Gruber, K., Jogl, G., Wagner, U. G., Bothe, H., Buckel, W., and Kratky, C. 1999, Structure of coenzyme Bj2 dependent enzyme glutamate mutase from Clostridium cochlearium. Structure 7 8919902. 

Gruber, K., Reitzer, R., Kratky, C. (2001) Radical shuttling in a protein Ribose pseudorotation controls alkyl-radical transfer in the coenzyme B12 dependent enzyme glutamate mutase, Angew. Chem. Int. Ed. Engl. 40, 3377-3380. 

The enzyme glutamate mutase catalyzes such a rearrangement in which carboxylate (C02 ) and a neighboring hydrogen of glutamate exchange their points of attachment. 

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

The rearrangement described above by Barker using the coenzyme B12-dependent enzyme glutamate mutase is a most remarkable reaction. Until very recently, no analogous chemical reaction was known. In fact, elucidation of the structure of the coenzyme form of vitamin B12 did not clarify its mechanism. Beside this transformation, nine distinct enzymatic reactions requiring coenzyme B12 as cofactor are known. Most of which are without precedent in terms of organic reactions. They are listed in Fig. 6.11. In choosing vitamin B12 derivatives as coenzymes, enzymes appear to have reached a peak of chemical sophistication which would be difficult to mimic by the chemist. 

This cobalamin-dependent enzyme [EC 5.4.99.1], also known as glutamate mutase or methylaspartate mutase, catalyzes the interconversion of L-r/ireo-3-methylaspar-tate and L-glutamate. 

Recently, EPR spectroscopy with 2H- and 13C-labeled glutamates as substrates for glutamate mutase permitted identification of a 4-glutamyl radical as a probable intermediate for that enzyme.402/402a/b... 

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... 

Both methylmalonyl-CoA mutase and glutamate mutase share strikingly similar global folds (Figure 8), even though sequence similarity is limited to the small a/ 3 domain and their quaternary structures are quite different. The icatalytici domains of both enzymes take the form of a ( a/p)s TIM-barrel the Ca atoms of the two structures can be superimposed with an r.m.s. deviation of only 2 (Reitzer et al., 1999). However, the active site residues of these enzymes (other than those involved in binding the lower face of the coenzyme) do not seem to be conserved and the substrates are bound very differently. 

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

Chen, H. P., and Marsh, E. N. G., 1997, How enzymes control the reactivity of adenosylcobal-amin effect on coenzyme binding and catalysis of mutations in the conserved histidine-aspartate pair of glutamate mutase. Biochemistry 36 788497889. 

Holloway, D. E., and Marsh, E. N. G., 1994, Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum. Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme. J. Biol. Chem. 269 20425920430. 

About 10 coenzyme B -dependent enzymes are now known (reviewed in References 13,14, and 76 see Table 1) four carbon skeleton mutases (methylmalonyl-CoA mutase (MMCM), glutamate mutase (GM), methylene glu-tarate mutase (MGM), isobutyryl-CoA mutase (ICM) ), diol dehydratase (DD), glycerol dehydratase, ethanol-amine anunonia lyase (EAL), two amino mutases, and Bi2-dependent ribonucleotide reductase. The coenzyme Bi2-dependent enzymes are unevenly distributed in the living world, and MMCM is the only enzyme that is also indispensable in human metabolism. ... 

Glutamate mutase. GM from Clostridium tetanomor-phum was the first enzyme discovered (around 1960) to be dependent upon a Bn-coenzyme. GM catalyzes the... 

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... 

The first common step in AdoCbl-dependent readions is homolytic cleavage of the cobalt-carbon bond to generate a radical pair, cob(ii)alamin and the carbon-centered dAdo radical (Scheme 19.3). This reaction experiences a 10 -fold rate enhancement in B12 enzymes [14, 15] in the presence of substrate, and the mechanism for this rate acceleration has been the subject of extensive scrutiny. Thus, in methylmalonyl-CoA mutase and in glutamate mutase, little if any destabilization of the cobalt-carbon bond is observed in the reactant state, as revealed by resonance Raman spectroscopy [16, 17], and the intrinsic substrate binding is utilized to labilize the bond. In contrast, approximately half of the destabilization of the cobalt-carbon bond in diol dehydratase is expressed in the reactant state. This re-... 

In glutamate mutase [43], the forward and reverse steady-state deuterium (kn/ko of 3.9 forward and 6.3 reverse) and tritium kn/kj of 21 forward and 19 reverse) kinetic isotope effects are both suppressed. However large deuterium isotope effects of 28 and 35 in the forward and reverse directions respectively have been observed for cob(ii)alamin formation under pre-steady-state conditions. These large kinetic isotope effects suggest that quantum mechanical tunneling also dominates this enzyme reaction. 

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