Inhibition of phenylalanine hydroxylase by p-chlorophenylalanine; Dependence on cofactor structure

The reported discrepancy between the in vitro and in vivo properties of p-chlorophenylalanine as an inhibitor of phenylalanine hydroxylase (E.C.1.14. 3.1) was investigated. It was demonstrated that the lack of inhibition, in vitro, was not due to (1) non-physiological pH or temperature of the in vitro assay system, (2) inhibition by m-chlorotyrosine, a product of the enzymatic hydroxylation of p-chlorophenylalanine, or (3) a slow irreversible reaction of p-chlorophenylalanine with enzyme. However, when the inhibitory properties of p-chlorophenylalanine were determined using the natural cofactor, tetrahydrobiopterin, instead of the pseudocofactor 6,7-dimethyltetrahydropterin, which had been utilized in the reported in vitro studies, it was shown that p-chlorophenylalanine is a potent inhibitor of the enzymatic hydroxylation of phenylalanine. The apparent K i is 0.03mM with tetrahydobiopterin as cofactor, compared to 1.5mM with 6.7-dimethyltetrahydropterin. The dependence of the inhibitory properties of an aromatic amino acid analog on the structure of the cofactor may be a general phenomenon with all tetrahydrobiopterin dependent aromatic amino acid hydroxylases.