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-dependent 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 K(M) 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-methyltetrahydropterin 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.