title: Mutational analysis of catecholamine binding in tyrosine hydroxylase.
abstract: Tyrosine hydroxylase (TH) performs the first and rate-limiting step in the synthesis of catecholamines, which feed back to regulate the enzyme by irreversibly binding to a high-affinity site and inhibiting TH activity. Phosphorylation of Ser40 relieves this inhibition by allowing dissociation of catecholamine. We have recently documented the existence of a low-affinity catecholamine binding which is dissociable, is not abolished by phosphorylation, and inhibits TH by competing with the essential cofactor, tetrahydrobiopterin. Here, we have substituted a number of active site residues to determine the structural nature of the low- and high-affinity sites. E332D and Y371F increased the IC(50) of dopamine for the low-affinity site 10-fold and 7 0-fold, respectively, in phosphorylated TH, indicating dramatic reductions in affinity. Only 2-4-fold increases in IC(50) were measured in the nonphosphorylated forms of E332D and Y371F and also in L294A and F300Y. This suggests that while the magnitude of low-affinity site inhibition in wild-type TH remains the same upon TH phosphorylation as previously shown, the active site structure changes to place greater importance on E332 and Y371. Changes to high affinity binding were also measured, including a loss of competition with tetrahydrobiopterin for E332D, A297L, and Y371F and a decreased ability to inhibit catalysis (V(max)) for A297L and Y371F. The common roles of E332 and Y371 indicate that the low- and high-affinity catecholamine binding sites are colocalized in the active site, but due to simultaneous binding, may exist in separate monomers of the TH tetramer.
authors.affiliation: The School of Biomedical Sciences and Pharmacy and The Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, New South Wales 2308, Australia.
authors.forename: Gabrielle D
authors.initials: GD
authors.lastname: Briggs
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Gordon
authors.forename: Phillip W
authors.initials: PW
authors.lastname: Dickson
mesh_terms: D020134:Catalytic Domain; D002395:Catecholamines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D011485:Protein Binding; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002395:Catecholamines; D014446:Tyrosine 3-Monooxygenase