Site-directed mutants of charged residues in the active site of tyrosine hydroxylase

The active site of tyrosine hydroxylase consists of a hydrophobic cleft with an iron atom near the bottom. Within the cleft are several charged residues which are conserved across the family of pterin-ependent hydroxylases. We have studied four of these residues, glutamates 326 and 332, aspartate 328, and arginine 316 in tyrosine hydroxylase, by site-directed substitution with alternate amino acid residues. Replacement of arginine 316 with lysine results in a protein with a K-tyr value that is at least 400-fold greater and a V/K-tyr value that is 4000-fold lower than those found: in the wild-type enzyme; substitution with alanine, serine, or glutamine yields insoluble enzyme. Arginine 316 is therefore critical for the binding of tyrosine. Replacement of glutamate 326; with alanine has no effect On the KM value for tyrosine and results in a 2-fold increase in the K-M value for tetrahydropterin. The V-max for DOPA production is reduced 9-fold, and the V-max for dihydropterin formation is reduced 4-fold. These data suggest that glutamate 326 is not directly involved in catalysis. Replacement of aspartate 328 with serine results in a 26-fold higher K-M value for tyrosine, a 8-fold lower V-max for dihydropterin formation, and a 13-fold lower V-max for DOPA formation. These data suggest that aspartate 328 has a role in tyrosine binding. Replacement of glutamate 332 with alanine results in a 10-fold higher K-M value for 6-methyltetrahydrapterin with no change in the K-M value for tyrosine, a 125-fold lower V-max for DOPA formation, and an only 3.3-fold lower V-max for tetrahydropterin oxidation. These data suggest that glutamate 332 is required for productive tetrahydropterin binding.