PURIFICATION AND PROPERTIES OF 2-AMINOETHYLPHOSPHONATE - PYRUVATE AMINOTRANSFERASE FROM PSEUDOMONAS-AERUGINOSA
PURIFICATION AND PROPERTIES OF 2-AMINOETHYLPHOSPHONATE - PYRUVATE AMINOTRANSFERASE FROM PSEUDOMONAS-AERUGINOSA
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DOI：
10.1111/j.1432-1033.1983.tb07436.x
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
CASSAIGNE, A
中科院分区：
文献类型：
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作者：
DUMORA, C;LACOSTE, AM;CASSAIGNE, A
从铜绿假单胞菌细胞提取液中纯化得到纯度为15%的2-氨基乙基膦酸氨基转移酶。凝胶过滤测得该酶的相对分子质量为65,000。+-。2000年。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法测得分子量为16,500。+-。1000，表明该蛋白为四聚体模型。在280、335和415 nm处有最大吸收峰，这是一种依赖于吡哆醛的酶的特征，其吸收峰为4摩尔的5‘-磷酸吡哆醛/摩尔酶。该氨基转移酶催化2-氨基乙基膦(纤毛酸)的氨基转移到丙酮酸，生成2-膦乙醛脱氢染料和丙氨酸。酶的最适pH在8.5-9之间，酶活从30度提高。到50度。C组。该反应符合Michaelis-Menten动力学，纤毛酸和丙酮酸的Km值分别为3.85和3.5 mm。该酶表现出很高的特异性，因为纤毛酸和丙酮酸分别是唯一的氨基供体和受体。与它们的α-氨基衍生物相比，甲基、乙基和丙基膦酸是纤毛素更好的竞争对手。3-氨基丙基膦酸脂是纤毛素的高级同系物，既不是底物，也不是抑制物。羧基试剂和HgCl2对酶活性有显著影响。2-氨基乙基膦的转氨化是导致其C.sbd.p键断裂的两步途径的第一步。
2-Aminoethylphosphonate aminotransferase was purified to homogeneity with a yeild of 15% from cell extracts of P. aeruginosa. The MW of the enzyme was estimated by gel filtration to be 65,000 .+-. 2000. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a MW of 16,500 .+-. 1000, suggesting a tetrameric model for this protein. The absorption spectrum exhibits maxima at 280, 335 and 415 nm, which are characteristic of a pyridoxal-phosphate-dependent enzyme: 4 mol of pyridoxal 5''-phosphate/mol of enzyme, were found. This aminotransferase catalyzes the transfer of the amino group of 2-aminoethylphosphonate (ciliatine) to pyruvate to give 2-phosphonoacetaldehdye and alanine. A pH optimum between 8.5-9 and an activity increasing from 30.degree. to 50.degree. C were observed. The reaction follows Michaelis-Menten kinetics, with Km values of 3.85 and 3.5 mM for ciliatine and pyruvate, respectively. This enzyme shows a very high specificity, since ciliatine and pyruvate are the only amino donor and acceptor, respectively. Methyl, ethyl and propylphosphonic acids are better competitors towards ciliatine than their .alpha.-amino derivatives. 3-Aminopropylphosphonate, the higher homologue of ciliatine, is recognized neither as a substrate nor as an inhibitor. The enzyme activity is significantly affected by carbonyl reagents and by HgCl2. Transamination of 2-aminoethylphosphonate is the 1st step of a double-step pathway which leads of the cleavage of its C.sbd.P bond.