Mutagenesis of the regulatory domain of phenylalanine hydroxylase

The regulatory domain of phenylalanine hydroxylase (PAH. EC 1.14.16.1) cons ists of more than 100 amino acids at the N terminus, the removal of which s ignificantly activates the enzyme. To study the regulatory properties contr olled by the N terminus, a series of truncations and site-specific mutation s were made in this region of rat PAH. These enzymes were expressed highly in Escherichia coli and purified through a pterin-conjugated Sepharose affi nity column. The removal of the first 26 amino acids of the N terminus incr eased the activity by about 20-fold, but removal of the first 15 amino acid s increased the activity by only 2-fold. Replacing serine-29 of rat PAH wit h cysteine from the same site of human PAH increased the activity by more t han 4-fold. Mutation of serine to other amino acids with varying side chain s: alanine, methionine, leucine, aspartic acid, asparagine, and arginine al so resulted in significant activation. indicating a serine-specific inhibit ory effect. But these site-specific mutants showed 30-40% lower activity wh en assayed with 6-methyl-5,6,7,8-tetrahydropterin, Stimulation of hydroxyla se activity by preincubation of the enzyme with phenylalanine was inversely proportional to the activation state of all these mutants. Combined with r ecent crystal structures of PAH [Kobe, B. et al. (1999) Nat. Struct. Biol, 6, 442-448; and Erlandsen, H., Bjorgo, E., Flatmark, T. & Stevens, R. C, (2 000) Biochemistry 39, 2208-2217], these data suggest that residues 16-26 ha ve a controlling regulatory effect on the activity by interaction with the dihydroxypropyl side chain of (6R)-5,6,7,8-tetrahydrobiopterin. The serine/ cysteine switch explains the difference in regulatory properties between hu man and rat PAH. The IU terminus as a whole is important for maintaining ra t PAH in an optimum catalytic conformation.