Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri.
The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin. This preparation could be reactivated to 95-100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5'-3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme. Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose. The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.
Michel
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C
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Federal Republic of Germany.
Albracht
S P
SP
Buckel
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W
D003013:Clostridium / Q000201:enzymology*; D003038:Cobamides / Q000032:analysis*; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D019751:Intramolecular Transferases; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013053:Spectrophotometry; D014805:Vitamin B 12 / Q000032:analysis*
D003038:Cobamides; D007535:Isomerases; D019751:Intramolecular Transferases; C047997:2-methyleneglutarate mutase; C000913:cobamamide; D014805:Vitamin B 12; D004229:Dithiothreitol