On the role of two different cobalt(II) species in coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri.
Purified 2-methyleneglutarate mutase from Clostridium barkeri contains adenosylcobalamin (coenzyme B12) and varying amounts of oxygen-stable cob(II)alamin. The content of the latter was estimated by EPR spectroscopy at 6-11% of the total cobalamin (2-4 mol/mol enzyme). Tryptic digestion of the enzyme liberated the prosthetic groups, cob(II)alamin being oxidized by air to aquocobalamin. HPLC analysis of the released cobamides from several preparations revealed > 90% adenosylcobalamin and < 10% aquocobalamin. Treatment of active 2-methyleneglutarate mutase with 8M urea followed by gelfiltration yielded an inactive enzyme from which 50% of the adenosylcobalamin and up to 70% of the cob(II)alamin was removed. Addition of adenosylcobalamin to the urea-treated enzyme resulted in complete reactivation, but the content of cob(II)alamin was not increased. These data suggest that the oxygen-stable cob(II)alamin is not involved in catalysis. In the presence of the competitive inhibitor itaconate (methylenesuccinate, Ki = 0.7mM), an alteration of the UV/visible spectrum at 470 nm as well as a new line in the EPR spectrum of the enzyme (around g = 2.1) was observed. The results indicate the formation of an unusual, oxygen sensitive Co(II) species during catalysis. The EPR signal of the oxygen-stable cob(II)alamin (gx,y = 2.24) remained unchanged under those conditions.
Zelder
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Laboratorium für Mikrobiologie im Fachbereich Biologie, Phillipps-Universität Marburg.
Buckel
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W
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology*; D003035:Cobalt / Q000032:analysis / Q000737:chemistry*; D003038:Cobamides / Q000032:analysis / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D019751:Intramolecular Transferases; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D003038:Cobamides; D003035:Cobalt; D007535:Isomerases; D019751:Intramolecular Transferases; C047997:2-methyleneglutarate mutase; C000913:cobamamide