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.
Leutbecher
U
U
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Federal Republic of Germany.
Böcher
R
R
Linder
D
D
Buckel
W
W
D000591:Amino Acid Isomerases / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology*; D003038:Cobamides / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000494:pharmacology; D019751:Intramolecular Transferases; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
D000591:Amino Acid Isomerases; D001426:Bacterial Proteins; D003038:Cobamides; D005971:Glutamates; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000731191:component S, glutamate mutase protein, Bacteria; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)