Ganelin V L, Razlogova I O, Petiushenko R M, Chernyshev A I, Esipov S E
Biokhimiia. 1978 Dec;43(12):2154-62.
An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.
一种催化某些具有3'-羟基的氨基糖苷类抗生素磷酸化的氨基糖苷-3'-磷酸转移酶I，已通过新霉素-琼脂糖亲和柱层析和Sephadex G-100凝胶过滤，从耐氨基糖苷类菌株大肠杆菌182的细胞中纯化得到。通过核磁共振、薄层层析和化学表征，分离并鉴定了卡那霉素A酶促磷酸化的产物为卡那霉素-3'-磷酸。研究了该酶的动力学性质。最适pH值在7.8至8.0之间；卡那霉素、新霉素和巴龙霉素的[S]0.5值为2×10⁻⁵ M，活化能为15.9千卡/摩尔。该酶的活性需要二价阳离子，Mg²⁺最为有效。不含3'-羟基的相对氨基糖苷类抗生素是该酶活性的竞争性抑制剂，其Ki值接近[S]0.5。
