Functional analysis, using in vitro mutagenesis, of amino acids located inthe phenylalanine hydroxylase active site

The 3-dimensionaI structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active si te cleft with the majority of these having hydrophobic side-chains includin g several with aromatic side chains. Here we have analyzed the effect on ra t PAH enzyme kinetics of in vitro mutagenesis of a number of these amino ac ids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caus ed a significant decrease in enzyme activity but no change in the K-m for s ubstrate or cofactor. me conclude that these aromatic residues are essentia l for activity but are not significantly involved in binding of the substra te or cofactor. in contrast the PAH mutant, S349T, showed an 18-fold increa se in K-m for phenylalanine, showing the first functional evidence that thi s residue was binding at or near the phenylalanine binding site. This confi rms the recently published model for the binding of phenylalanine to the PA H active site that postulated S349 interacts with the amino group on the ma in chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydro xylase, which had no effect on substrate K-m, showing that while the archit ecture of the two active sites are very similar the amino acids that bind t o the respective substrates are different. (C) 2000 Academic Press.