A general coupled spectrophotometric assay for decarboxylases.

A rapid, continuous spectrophotometric method has been developed for the assay of decarboxylases. The assay uses a coupled enzyme system in which liberated CO2 is reacted with phosphoenolpyruvate and phosphoenolpyruvate carboxylase to form oxaloacetate, which in turn is reduced by malate dehydrogenase to L-malate concomitantly with the oxidation of NADH to NAD. The resultant decrease in absorbance at 340 nm accurately reflects the activity of the decarboxylase.

Separation by high-performance liquid chromatography of alpha- and beta-amino acids: application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase.

Several pairs of alpha- and beta-amino acids were well separated as the ortho-phthalaldehyde derivatives by reversed phase HPLC. These included alpha- and beta-lysine, leucine, tyrosine, serine, aminoisobutyric acid, and beta-hydroxyvaline and its positional isomer. The separation was applicable to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase.

Substrate stereochemistry of 2-methyl-branched-chain acyl-CoA dehydrogenase: elimination of one hydrogen each from (pro-2S)-methyl and alpha-methine of isobutyryl-CoA.

Dehydrogenation of (2S)-[3-13C]isobutyryl-CoA was carried out in vitro using 2-methyl-branched-chain acyl-CoA dehydrogenase purified from rat liver mitochondria. The product was subsequently hydrated by the addition of bovine crotonase. The resulting 3-hydroxyisobutyric acid was predominantly enriched with 13C at the beta-hydroxy position as determined as a methyl ester using electron ionization-gas chromatography-mass spectroscopy.