Properties of D-arabinose isomerase purified from two strains of Escherichia coli.
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 x 10(-13) sec for the E. coli K-12 enzyme and 14.3 x 10(-13) sec for the E. coli B/r enzyme. The molecular weight, determined by high-speed sedimentation equilibrium, was 3.55 +/- 0.06 x 10(5) for the E. coli K-12 enzyme and 3.42 +/- 0.04 x 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 x 10(-1)m for d-arabinose and 4.5 x 10(-2)m for l-fucose; with the E. coli B/r enzyme, the K(m) was 1.7 x 10(-1)m for d-arabinose and 4.2 x 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 pH 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 x 10(4) for the enzyme from E. coli K-12 and 8.46 +/- 0.1 x 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.
D000596:Amino Acids / Q000032:analysis; D001089:Arabinose / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D002474:Cell-Free System; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003035:Cobalt / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005643:Fucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D013045:Species Specificity; D013237:Stereoisomerism; D013402:Sugar Alcohols / Q000494:pharmacology; D014461:Ultracentrifugation
D000596:Amino Acids; D013402:Sugar Alcohols; D005643:Fucose; D003035:Cobalt; D008345:Manganese; D001089:Arabinose; D002238:Carbohydrate Epimerases