Properties of d -Arabinose Isomerase Purified from Two Strains of Escherichia coli

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Abstract
D -Arabinose isomerase (EC 5.3.1.3) has been isolated from l -fucose-induced cultures of Escherichia coli K-12 and d -arabinose-induced cultures of E. coli B/r. Both enzymes were homogeneous in an ultracentrifuge and migrated as single bands upon disc electrophoresis in acrylamide gels. The s 20,w was 14.5 × 10 −13 sec for the E. coli K-12 enzyme and 14.3 × 10 −13 sec for the E. coli B/r enzyme. The molecular weight, determined by high-speed sedimentation equilibrium, was 3.55 ± 0.06 × 10 5 for the E. coli K-12 enzyme and 3.42 ± 0.04 × 10 5 for the enzyme isolated from E. coli B/r. Both enzyme preparations were active wth l -fucose or d -arabinose as substrates and showed no activity on any of the other aldopentoses or aldohexoses tested. With the E. coli K-12 enzyme, the K m was 2.8 × 10 −1 m for d -arabinose and 4.5 × 10 −2 m for l -fucose; with the E. coli B/r enzyme, the K m was 1.7 × 10 −1 m for d -arabinose and 4.2 × 10 −2 m for l -fucose. Both enzymes were inhibited by several of the polyalcohols tested, ribitol, l -arabitol, and dulcitol being the strongest. Both enzymes exhibited a broad plateau of optimal catalytic activity in the alkaline range. Both enzymes were stimulated by the presence of Mn 2+ or Co 2+ ions, but were strongly inhibited by the presence of Cd 2+ ions. Both enzymes were precipitated by antisera prepared against either enzyme preparation. The amino acid composition for both proteins has been determined; a striking similarity has been detected. Both enzymes could be dissociated, by protonation at p H 2 or by dialysis against buffer containing 8 m urea, into subunits that were homogeneous in an ultracentrifuge and migrated as single bands on disc electrophoresis in acrylamide gels containing urea. The molecular weight of the subunit, determined by high-speed sedimentation equilibrium, was 9.09 ± 0.2 × 10 4 for the enzyme from E. coli K-12 and 8.46 ± 0.1 × 10 4 for the enzyme from E. coli B/r. On the basis of biophysical studies, both isomerases appear to be oligomeric proteins consisting of four identical subunits.