Purification and properties of 2-aminoethylphosphonate:pyruvate aminotransferase from Pseudomonas aeruginosa.
2-Aminoethylphosphonate aminotransferase has been purified to homogeneity with a yield of 15% from cell extracts of Pseudomonas aeruginosa. The molecular weight of the enzyme was estimated by gel filtration to be 65000 +/- 2000. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 16500 +/- 1000, suggesting a tetrameric model for this protein. The absorption spectrum exhibits maxima at 280 nm, 335 nm and 415 nm which are characteristic of a pyridoxal-phosphate-dependent enzyme: 4 mol of pyridoxal 5'-phosphate/mol of enzyme have been found. This aminotransferase catalyzes the transfer of the amino group of 2-aminoethylphosphonate (ciliatine) to pyruvate to give 2-phosphonoacetaldehyde and alanine. A pH optimum between 8.5-9 and an activity increasing from 30 degrees C to 50 degrees C have been observed. The reaction follows Michaelis-Menten kinetics with Km values of 3.85 mM 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 first step of a double-step pathway which leads to the cleavage of its C-P bond.[1]References
- Purification and properties of 2-aminoethylphosphonate:pyruvate aminotransferase from Pseudomonas aeruginosa. Dumora, C., Lacoste, A.M., Cassaigne, A. Eur. J. Biochem. (1983) [Pubmed]
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