Mutation of serine 395 of tyrosine hydroxylase decouples oxygen-oxygen bond cleavage and tyrosine hydroxylation

Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conser ved in all three members of the family of pterin-dependent hydroxylases, ph enylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S 395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respe ctively. Both the S395A and S396A enzymes bind a stoichiometric amount of i ron and exhibit wild-type spectra when complexed with dopamine. The K-M val ues for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are una ffected by replacement of either residue with alanine. Although the V-max v alue for tyrosine hydroxylation by the S395A enzyme is decreased by 2 order s of magnitude, the V-max value for tetrahydropterin oxidation by either th e S395A or the S396A enzyme is unchanged from the wild-type value. With bot h mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required f or amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxy lation.