Ethanolamine Deaminase, a Cobamide Coenzyme-dependent Enzyme: II. PHYSICAL AND CHEMICAL PROPERTIES AND INTERACTION WITH COBAMIDES AND ETHANOLAMINE

Abstract

Abstract A purified preparation of clostridial ethanolamine deaminase was shown to be homogeneous by sedimentation in the ultracentrifuge and by disc gel electrophoresis. The enzyme has an s20,w of 14.5 S and a weight average molecular weight of 520,000 g. Treatment with 5 m guanidine hydrochloride results in dissociation of subunits with a weight average molecular weight of 51,000 g. Amino acid analysis of native and performic acid-oxidized deaminase indicated the presence of 3 moles of half-cystine residues per minimum molecular weight of 28,700 g. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) yielded only 1 mole of free sulfhydryl group per mole of native enzyme (520,000 g) but in the presence of sodium lauryl sulfate 15 sulfhydryl groups per molecule were titratable. The spectrum of the enzyme contains a prominent peak at 470 mµ, with other peaks at 357, 410, and 530 mµ. The chromophore was resolved from the enzyme by treatment with acid-ammonium sulfate. On resolution, the 470 mµ peak of the enzyme disappeared and the dissociated chromophore had a spectrum similar to that of hydroxocobalamin and related cobamides. The chromophore was identified as an α-(adenylyl)cobamide by paper chromatography and paper electrophoresis. Between 1.35 and 3.1 moles of cobamide were found per mole of native enzyme. On resolution, the specific activity of the deaminase, when tested in the presence of added cobamide coenzyme, increased an average of 38%. Hydroxocobalamin, cyanocobalamin, and methylcobalamin were shown to be irreversible inhibitors of ethanolamine deaminase. Incubation of resolved enzyme with varying amounts of hydroxocobalamin resulted in complete inhibition of the enzyme after the binding of 2 to 3 moles of cobamide per mole of enzyme. The enzyme was able to bind about 7 moles of hydroxocobalamin per mole. In both the presence and absence of ethanolamine, incubation of the enzyme resulted in the slow appearance of a spectral peak at 352 mµ. Oxidation and Schmidt degradation of acetaldehyde produced by the enzymatic deamination of 1-14C-ethanolamine hydrochloride showed that the aldehyde carbon of the acetaldehyde is derived from the carbinol carbon of ethanolamine.