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Investigations into the mechanism of the coenzyme B12 dependent reaction catalyzed by glutamate mutase from Clostridium cochlearium
Aims of this study were the search for inhibitors of the coenzyme B12-dependent glutamate mutase and for insight into the first step of its catalytic mechanism, the homolytic cleavage of the cobalt-carbon bond. Glutamate mutase is composed of two separately isolated protein components S and E2, whic...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2012
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| Zusammenfassung: | Aims of this study were the search for inhibitors of the coenzyme B12-dependent glutamate mutase and for insight into the first step of its catalytic mechanism, the homolytic cleavage of the cobalt-carbon bond. Glutamate mutase is composed of two separately isolated protein components S and E2, which in the presence of coenzyme B12 assemble to the active holo-glutamate mutase E2S2-B12 that catalyzes the reversible conversion of (S)-glutamate to (2S,3S)-3-methylaspartate. This reaction has been coupled with methylaspartase, which deaminates (2S,3S)-3-methylaspartate to mesaconate absorbing at 240 nm, to allow activity assays for glutamate mutase by UV-spectrophotometry. As potential inhibitors, compounds with sp2-centers and structural analogies to the intermediate radicals in the proposed mechanism were selected. Analogues to the 4-glutamyl radical were (E)- and (Z)-glutaconates, whereas analogues to the (2S,3S)-3-methyleneaspartate radical included itaconate, buta-1,3-diene-2,3-dicarboxylate, fumarate, maleate and mesaconate. Because all these compounds inhibited the auxiliary enzyme methylaspartase, glutamate mutase was incubated with these compounds for a certain time, followed by gelfitration on Sephadex G25. The residual activity of the inactivator-free enzyme was then determined by the coupled assay described above, whereby unexpectedly fumarate, maleate and mesaconate caused inactivation of the mutase. To check whether the other compounds acted as reversible inhibitors, a new assay with (2S,3S)-3-methylaspartate and pyruvate as substrates involving glutamate-pyruvate aminotransferase and the NADH-dependent (R)-2-hydroxyglutarate dehydrogenase was developed. Application of this assay showed that 2.5 mM itaconate and 8 mM (E)-glutaconate inhibited glutamate mutase in the presence of 200 mM (2S,3S)-3-methylaspartate by 50%. Furthermore, the kinetic constants of (2S,3S)-3-methylaspartate in the reaction of glutamate mutase were determined as Km= 7 ± 0.07 mM, kcat= 0.54 ± 0.06 s-1and kcatKm-1= 77 s-1M-1. Together with the kinetic constants of (S)-glutamate determined with the methylaspartase assay (Km = 2.25 ± 0.03 mM, kcat = 2.85 ± 0.5 s-1 and kcatKm-1 = 1.3 × 10-3 s-1M-1), an equilibrium constant of Keq = [glutamate] × [methylaspartate]-1 = 16 was calculated by the Briggs-Haldane equation close to that described in the literature (Keq = 12).
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The mutL gene from Clostridium tetanomorphum is located between the structural genes of glutamate mutase. We speculate that MutL acts as chaperone, which removes cob(II)alamin from inactive glutamate mutase complexes in an ATP dependent manner. The liberated components E2 and S recombine with coenzyme B12 to form a new active enzyme. To check this hypothesis, mutL was successful cloned on pASG-IBA3 and pASG-IBA 5 expression vectors via the pre-entry vector IBA-20. The MutL chaperone was produced in E. coli Rossetta in good yields |
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| DOI: | 10.17192/z2013.0109 |