Boulter J R, Gielow W O
J Bacteriol. 1973 Feb;113(2):687-96. doi: 10.1128/jb.113.2.687-696.1973.
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.
D-阿拉伯糖异构酶（EC 5.3.1.3）已从大肠杆菌K-12的L-岩藻糖诱导培养物和大肠杆菌B/r的D-阿拉伯糖诱导培养物中分离出来。两种酶在超速离心机中均呈均一状态，在聚丙烯酰胺凝胶圆盘电泳中迁移时呈现单一条带。大肠杆菌K-12酶的沉降系数s(20,w)为14.5×10⁻¹³秒，大肠杆菌B/r酶的沉降系数为14.3×10⁻¹³秒。通过高速沉降平衡法测定，大肠杆菌K-12酶的分子量为3.55±0.06×10⁵，从大肠杆菌B/r分离出的酶的分子量为3.42±0.04×10⁵。两种酶制剂均以L-岩藻糖或D-阿拉伯糖为底物时具有活性，而对所测试的其他任何戊醛糖或己醛糖均无活性。对于大肠杆菌K-12酶，D-阿拉伯糖的米氏常数（K(m)）为2.8×10⁻¹摩尔/升，L-岩藻糖的K(m)为4.5×10⁻²摩尔/升；对于大肠杆菌B/r酶，D-阿拉伯糖 的K(m)为1.7×10⁻¹摩尔/升，L-岩藻糖的K(m)为4.2×10⁻²摩尔/升。所测试的几种多元醇对两种酶均有抑制作用，其中核糖醇、L-阿拉伯糖醇和卫矛醇的抑制作用最强。两种酶在碱性范围内均表现出较宽的最佳催化活性平台。Mn(2+)或Co(2+)离子的存在可刺激两种酶的活性，但Cd(2+)离子的存在则强烈抑制其活性。两种酶制剂的抗血清均可使两种酶沉淀。已测定了两种蛋白质的氨基酸组成；发现它们具有显著的相似性。通过在pH 2下质子化或用含8摩尔/升尿素的缓冲液透析，两种酶均可解离成在超速离心机中呈均一状态且在含尿素的聚丙烯酰胺凝胶圆盘电泳中迁移时呈现单一条带的亚基。通过高速沉降平衡法测定，大肠杆菌K-12酶亚基的分子量为9.09±0.2×10⁴，大肠杆菌B/r酶亚基的分子量为8.46±0.1×10⁴。基于生物物理研究，两种异构酶似乎都是由四个相同亚基组成的寡聚蛋白。
