Synthesis of [3H1]-2-Aminohexanoid Acid as a Substrate for Phenylalanine Hydroxylase
The amino acid 2-aminohexanoic acid (norleucine) was synthesized to be used as an artificial substrate for the enzyme phenylalanine hydroxylase (PAH). This enzyme catalyzes the conversion of phenylalanine to tyrosine. which is studied in relation to the disease phenylketonuria. The mechanism by which phenylalanine is converted to tyrosine. however. is unknown. Norleucine is known to be hydroxylated in the E-position by PAH. It was labeled with tritium at the site of hydroxylation. and subjected to an enzyme assay in order to determine whether hydrogen is abstracted by PAH. [3H1]norleucine was synthesized and its radioactivity was counted at 5710 counts per minute per milligram norleucine. After incubation with the components of the hydroxylating system. the radioactivity of the enzyme was counted at 26 cpm. These results are inconclusive as to whether hydrogen is abstracted by phenylalanine hydroxylase due to insufficient tritium incorporation into the amino acid. This low radioactive count registered by the enzyme may be the result of several factors. but does not necessarily imply that no hydrogen was abstracted. Given the low count produced by the enzyme. in order to determine whether an abstraction has occurred. it would be necessary to use more sensitive counting techniques.