Enzymatic determination of L-phenylalanine and phenylpyruvate with L-phenylalanine dehydrogenase.
An enzymatic method is described for the determination of L-phenylalanine or phenylpyruvate using L-phenylalanine dehydrogenase. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine or the reductive amination of the 2-oxoacid, respectively. The stoichiometric coupling of the coenzyme allows a direct spectrophotometric assay of the substrate concentration. The equilibrium of the reaction favors L-phenylalanine formation; however, by measuring initial reaction velocities, the enzyme can be used for L-phenylalanine determination, too. Standard solutions of L-phenylalanine in the range of 10-300 microM and of phenylpyruvate (5-100 microM) show a linearity between the value for dENADH/min and the substrate concentration. Besides phenylalanine, the enzyme can convert tyrosine and methionine, and their oxoacids, respectively. The Km values of these substrates are higher. The influence of tyrosine on the determination of phenylalanine was studied and appeared tolerable for certain applications.
Hummel
W
W
Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Federal Republic of Germany.
Schütte
H
H
Kula
M R
MR
D000594:Amino Acid Oxidoreductases; D000596:Amino Acids / Q000032:analysis; D003641:Deamination; D010649:Phenylalanine / Q000032:analysis*; D010667:Phenylpyruvic Acids / Q000032:analysis*; D013053:Spectrophotometry; D013379:Substrate Specificity; D014443:Tyrosine / Q000032:analysis
D000596:Amino Acids; D010667:Phenylpyruvic Acids; D014443:Tyrosine; D010649:Phenylalanine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; C031606:phenylpyruvic acid