Stereochemistry of the reaction catalysed by 2-aminoethylphosphonate aminotransferase. A 1H-NMR study.
(R)- and (S)-2-amino[2-D1]ethylphosphonic acids ([2-D1]AEP) were synthesised to investigate the stereochemistry of the reaction catalysed by 2-aminoethlphosphonate aminotransferase from Pseudomonas aeruginosa. This enzyme catalyses the transfer of the amino group of AEP to pyruvate to produce 2-phosphonoacetaldehyde and alanine. The enzymic reaction proceeding through the abstraction of a proton from the Schiff-base complex formed between the enzyme-bound pyridoxal 5'-phosphate, and the substrate, was carried out in an aqueous buffer at pH 8.5; it was followed by high-field 1H-NMR measurements (500 MHz, H2O) on an AMX 500 Bruker spectrometer. The spectra, recorded with chiral (R)- or (S)-[2-D1]AEP, both showed the methylenic signal (3.0 ppm), whereas (S)-[2-D1]AEP gave the additional aldehydic signal (CHO, 9.6 ppm). These data clearly show that AEP-aminotransferase catalyses the abstraction of the pro-S hydrogen atom at the prochiral C2 carbon of AEP. Furthermore, careful timing of NMR measurements over a 2-hour period allows us to show the occurrence of an isotopic effect.
Département de Biochimie Médicale et Biologie Moléculaire, Université de Bordeaux II, France.
A M
AM
Lacoste
C
C
Dumora
L
L
Balas
F
F
Hammerschmidt
J
J
Vercauteren
D002384:Catalysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011522:Protons; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism
D011522:Protons; D000637:Transaminases; C038231:2-aminoethylphosphonate-pyruvate aminotransferase