title: Catalytic domain surface residues mediating catecholamine inhibition in tyrosine hydroxylase.
abstract: Tyrosine hydroxylase (TH) performs the rate-limiting step in catecholamine (CA) synthesis and is a tetramer composed of regulatory, catalytic and tetramerization domains. CAs inhibit TH by binding two sites in the active site; one with high affinity and one with low affinity. Only high affinity CA binding requires the regulatory domain, believed to interact with the catalytic domain in the presence of CA. Without a crystal structure of the regulatory domain, the specific areas involved in this process are largely undefined. It is not clear whether the regulatory domain-catalytic domain interaction is asymmetrical across the tetramer to produce the high and low affinity sites. To investigate this, pure dimeric TH was generated through double substitution of residues at the tetramerization interface and dimerization salt bridge (K170E/L480A). This was shown to be the core regulatory unit of TH for CA inhibition, possessing both high and low affinity CA binding sites, indicating that there is symmetry between dimers of the tetramer. We also examined possible regulatory domain-interacting regions on the catalytic domain that mediate high affinity CA binding. Using site-directed mutagenesis, A297, E362/E365 and S368 were shown to mediate high affinity dopamine inhibition through V(max) reduction and increasing the K(M) for the cofactor.
authors: 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: Gabrielle D
authors: GD
authors: Briggs
authors: Jesse
authors: J
authors: Bulley
authors: Phillip W
authors: PW
authors: Dickson
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D002395:Catecholamines / Q000494:pharmacology*; D002850:Chromatography, Gel; D004298:Dopamine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D012015:Reference Standards; D012984:Software; D013329:Structure-Activity Relationship; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002395:Catecholamines; D050505:Mutant Proteins; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine
keywords: catecholamine; end-point feedback inhibition; site-directed mutagenesis; tetrameric; tyrosine hydroxylase