A continuous coupled spectrophotometric assay for tyrosine aminotransferase activity with aromatic and other nonpolar amino acids.
A continuous assay for Escherichia coli tyrosine aminotransferase (TATase) that employs Lactobacillus delbrueckii ssp. bulgaricus hydroxyisocaproate dehydrogenase (HO-HxoDH) as a coupling enzyme is described. alpha-Keto acids, including those formed by TATase-catalyzed transamination of l-phenylalanine, l-tyrosine, l-tryptophan, l-methionine, and l-leucine, are converted to the corresponding alpha-hydroxy acids by the auxiliary enzyme. The concomitant reduction of NADH by this enzyme can be followed as a decrease in absorbance at 340 nm. Importantly, HO-HxoDHcatalyzed reduction of alpha-ketoglutarate (alpha-KG), a cosubstrate of TATase required to regenerate the pyridoxal-5'-phosphate cofactor of this enzyme from pyridoxamine-5'-phosphate, is a poor substrate and does not interfere with the assay. The kinetic parameters determined for the transamination of phenylalanine by TATase (kcat = 180 s-1, KM (L-Phe) = 0.56 mM, KM (alpha-KG) = 5 mM) with HO-HxoDH as a coupling enzyme are comparable to those reported in the literature, which were determined by direct monitoring of the formation of phenylpyruvate at 280 nm. This new assay offers the advantages of increased sensitivity and broad substrate specificity.
Luong
T N
TN
Department of Molecular and Cell Biology, University of California, Berkeley, 229 Stanley Hall #3206, Berkeley, California 94720-3206, USA.
Kirsch
J F
JF
D000429:Alcohol Oxidoreductases; D000596:Amino Acids / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007202:Indicators and Reagents; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013379:Substrate Specificity; D014444:Tyrosine Transaminase / Q000032:analysis* / Q000378:metabolism
D000596:Amino Acids; D007202:Indicators and Reagents; D000429:Alcohol Oxidoreductases; C047961:2-hydroxyisocaproate dehydrogenase; D014444:Tyrosine Transaminase