Fluorometric determination of phenylacetyl-CoA ligase from Pseudomonas putida: a very sensitive assay for a newly described enzyme.
Phenylacetyl-CoA ligase (AMP-forming) from Pseudomonas putida is a newly described enzyme (Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B. and Luengo, J.M. (1990) J. Biol. Chem. 265, 7084-7090) specifically involved in the catabolism of phenylacetic acid. This enzyme catalyzes the formation of phenylacetyl-CoA in the presence of ATP, CoA, Mg2+ and phenylacetic acid. A rapid method of assaying this enzyme in partially purified preparations has been developed by coupling this reaction with adenylate kinase, pyruvate kinase and kinase and lactate dehydrogenase. The rate of phenylacetyl-CoA formation was measured indirectly by monitoring fluorometrically the NADH oxidation at 340 nm (excitation at 340 nm and analysis of the emitted light at 465 nm). The advantage of this method of assay over others (colorimetric, HPLC and spectrophotometric) is discussed.
Rodríguez-Aparicio
L B
LB
Departamento Interfacultativo de Bioquimica y Biologia Molecular, Universidad de León, Spain.
Reglero
A
A
Martínez-Blanco
H
H
Luengo
J M
JM
D000105:Acetyl Coenzyme A / Q000096:biosynthesis; D000255:Adenosine Triphosphate / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000032:analysis* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D000105:Acetyl Coenzyme A; C037816:phenylacetyl-coenzyme A; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C023914:phenylacetate - CoA ligase