Identification of a paramagnetic species as an early intermediate in the coenzyme B12-dependent glutamate mutase reaction. A cob(II)amide?
Highly active and cobamide-free glutamate mutase was obtained from Clostridium cochlearium by a modification of the original purification procedure. After incubation of the enzyme with dithiothreitol, adenosylcobalamin (coenzyme B12) and the substrate (S)-glutamate, a paramagnetic species was observed by EPR-spectroscopy. The signal was maximal within 15 ms after mixing with glutamate. Different signals were detected after incubating the system with the competitive inhibitors (2S,4S)-4-fluoroglutamate or 2-methyleneglutarate instead of the substrate. The former developed with an at least 100-fold lower rate then the signal with glutamate. All three signals are probably due to low-spin cob(II)amide species with an extraordinary low gxy value as compared with cob(II)alamin.
Leutbecher
U
U
Laboratorium für Mikrobiologie, Philipps-Universität, Marburg, Germany.
Albracht
S P
SP
Buckel
W
W
D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001667:Binding, Competitive; D003038:Cobamides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D019751:Intramolecular Transferases; D013379:Substrate Specificity
D003038:Cobamides; D000591:Amino Acid Isomerases; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide