Substituting Tyr 138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation.

Mammalian phenylalanine hydroxylase (PAH) is a key enzyme in L-phenylalanine (L-Phe) metabolism and is active as a homotetramer. Biochemical and biophysical work has demonstrated that it cycles between two states with a variably low and a high activity, and that the substrate L-Phe is the key player in this transition. X-ray structures of the catalytic domain have shown mobility of a partially intrinsically disordered Tyr(138)-loop to the active site in the presence of L-Phe. The mechanism by which the loop dynamics are coupled to substrate binding at the active site in tetrameric PAH is not fully understood. We have here conducted functional studies of four Tyr(138) point mutants. A high linear correlation (r(2) = 0.99) was observed between their effects on the catalytic efficiency of the catalytic domain dimers and the corresponding effect on the catalytic efficiency of substrate-activated full-length tetramers. In the tetramers, a correlation (r(2) = 0.96) was also observed between the increase in catalytic efficiency (activation) and the global conformational change (surface plasmon resonance signal response) at the same L-Phe concentration. The new data support a similar functional importance of the Tyr(138)-loop in the catalytic domain and the full-length enzyme homotetramer.