title: The low affinity dopamine binding site on tyrosine hydroxylase: the role of the N-terminus and in situ regulation of enzyme activity.
abstract: Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.
authors.affiliation: School of Biomedical Sciences and The Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Gordon
authors.forename: Julianne K
authors.initials: JK
authors.lastname: Webb
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Shehadeh
authors.forename: Peter R
authors.initials: PR
authors.lastname: Dunkley
authors.forename: Phillip W
authors.initials: PW
authors.lastname: Dickson
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004298:Dopamine / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine