Glutamate mutase from Clostridium cochlearium. Purification, cobamide content and stereospecific inhibitors.
Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit. The enzyme proved to be very similar to that of C. tetanomorphum as described by Barker et al. [Barker, H. A., Rooze, V., Suzuki, F. & Iodice, A. A. (1964) J. Biol. Chem. 239, 3260-3266] but component E of C. cochlearium was more stable and led to the first pure preparation. The pink component E showed a cobamide-like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Co alpha-[alpha-(aden-9-yl)]-cob(II)amide. A typical cob(II)amide signal at g = 2.23 with hyperfine and superhyperfine splitting was observed by EPR spectroscopy. A cobamide content of about 0.43 mol/mol 50-kDa subunit was determined by cyanolysis. Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 microM). (2R,3RS)-3-Fluoroglutamate (Ki = 600 microM) was also inhibitory. The competitive inhibition by 2-methyleneglutarate (Ki = 400 microM) and (S)-3-methylitaconate (Ki = 100 microM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.