title: Identification of the 4-glutamyl radical as an intermediate in the carbon skeleton rearrangement catalyzed by coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.
abstract: A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.
authors.affiliation: Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universit-at, Marburg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Bothe
authors.forename: D J
authors.initials: DJ
authors.lastname: Darley
authors.forename: S P
authors.initials: SP
authors.lastname: Albracht
authors.forename: G J
authors.initials: GJ
authors.lastname: Gerfen
authors.forename: B T
authors.initials: BT
authors.lastname: Golding
authors.forename: W
authors.initials: W
authors.lastname: Buckel
mesh_terms: D002244:Carbon / Q000378:metabolism*; D002247:Carbon Isotopes; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D003038:Cobamides / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005971:Glutamates / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D003038:Cobamides; D005971:Glutamates; D005973:Glutamine; D002244:Carbon; D003903:Deuterium; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide