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 B-12) 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 g(xy) value as compared with cob(II)alamin.