title: Methylmalonyl-CoA mutase from Propionibacterium shermanii: characterization of the cobalamin-inhibited form and subunit-cofactor interactions studied by analytical ultracentrifugation.
abstract: A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidly converted into a yellow-brown compound which could be removed by dialysis. Enzyme activity could be recovered after removal of the denaturant, although surprisingly this did not depend on prior treatment with dithiothreitol. The interaction between the protein and inhibitor was investigated by using analytical ultracentrifugation under denaturing conditions. The sedimentation coefficient s20,w was measured in various concentrations of guanidine hydrochloride. A complicated picture emerged in which at low denaturant concentrations subunit dissociation, partial unfolding and aggregation occur, whereas at high concentration the protein behaves as a monodisperse species. No major differences in sedimentation were observed between the enzyme-cobalamin complex and the cobalamin-free enzyme, suggesting that the inhibitor does not significantly stabilize higher-order structure within the protein.
authors.affiliation: Department of Biochemistry, University of Cambridge, U.K.
authors.forename: E N
authors.initials: EN
authors.lastname: Marsh
authors.forename: S E
authors.initials: SE
authors.lastname: Harding
mesh_terms: D004229:Dithiothreitol / Q000494:pharmacology; D008765:Methylmalonyl-CoA Mutase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011424:Propionibacterium / Q000201:enzymology*; D011489:Protein Denaturation; D014461:Ultracentrifugation; D014805:Vitamin B 12 / Q000494:pharmacology*
chemical_list: D008765:Methylmalonyl-CoA Mutase; D014805:Vitamin B 12; D004229:Dithiothreitol