Purification and Properties of 2‐Aminoethylphosphonate: Pyruvate Aminotransferase from Pseudomonas aeruginusa

Abstract

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.