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 conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine 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, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for 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 hydroxylation.
Ellis
H R
HR
Department of Biochemistry, Texas A&M University, College Station, Texas 77843-2128, USA.
Daubner
S C
SC
Fitzpatrick
P F
PF
D000818:Animals; D006900:Hydroxylation; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism*; D017354:Point Mutation; D051381:Rats; D012694:Serine; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014443:Tyrosine; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D010100:Oxygen