Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: Aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation

The catalytic domains of the pterin-dependent enzymes phenylalanine hydroxy lase and tyrosine hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specif icity. seven residues in the active site of phenylalanine hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the correspo nding residues in tyrosine hydroxylase. Analysis of the effects of the muta tions on the isolated catalytic domain of phenylalanine hydroxylase identif ied three residues that contribute to the ability to hydroxylate tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-len gth phenylalanine hydroxylase and the complementary mutations into tyrosine hydroxylase. The steady-state kinetic parameters of the mutated enzymes sh owed that the identity of the residue in tyrosine hydroxylase at the positi on corresponding to position 379 of phenylalanine hydroxylase is critical f or dihydroxyphenylalanine formation. The relative specificity of tyrosine h ydroxylase for phenylalanine versus tyrosine, as measured by the (V/K-phe)/ (V/K-tyr) value, increased by 80000-fold in the D425V enzyme. However, muta tion of the corresponding valine 379 of phenylalanine hydroxylase to aspart ate was not sufficient to allow phenylalanine hydroxylase to form dihydroxy phenylalanine at rates comparable to that of tyrosine hydroxylase. The doub le mutant V379D/H264Q PheH was the most active at tyrosine hydroxylation, s howing a 3000-fold decrease in the (V/K-phe)/(V/K-tyr) value.