- Title
- Investigation of Catecholamine inhibition in Tyrosine Hydroxylase
- Creator
- Briggs, Gabrielle
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2013
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Tyrosine Hydroxylase (TH) performs the first and rate-limiting step in the synthesis of the catecholamines, dopamine, adrenaline and noradrenaline by converting tyrosine to DOPA. The catecholamines produce end-point feedback inhibition of TH, allowing for the control of cytosolic catecholamines in neurons and adrenal chromaffin cells. Catecholamines inhibit TH by binding to two sites; they bind irreversibly to the active site and will dissociate when the TH regulatory domain is phosphorylated at Ser40, and they bind to another site reversibly, regardless of Ser40 phosphorylation status. The location of this site had not been identified until now, and was shown to be located in the active site of TH, relying on E332 and Y371, which position catecholamine within the cofactor binding site, where it exerts its inhibition. These residues were found to play the same role in high affinity catecholamine binding, indicating that these two sites are colocalised. Since high and low affinity catecholamine binding occur simultaneously and produce stoichiometries of 1mol catecholamine per TH dimer for the high affinity site, and 1mol catecholamine per TH dimer for the low affinity site, it was hypothesised that these sites could not exist in the same active site, and may be spread across the tetramer of TH. Substitution of a residue at the tetramerisation interface (L480A), combined with a substitution of a residue involved in the dimerisation salt bridge (K170E) produced a pure dimeric species of TH that possessed both high and low affinity catecholamine binding sites. This indicated that the dimer is the core regulatory unit of TH for catecholamine inhibition. The high and low affinity catecholamine binding sites on TH were also explored by using another physiologically relevant catechol, salsolinol, which was shown to produce a more potent inhibition of phosphorylated TH than dopamine by producing a greater effect on cofactor binding. High affinity catecholamine binding was further explored by examining possible regulatory domain-interacting regions on the catalytic domain that are crucial for the irreversible binding of catecholamine. A297, D361 E362 and E365 were shown to mediate high affinity catecholamine binding, and, due to their location outside the active site, are likely to interact with the regulatory domain rather than catecholamine itself. This region of the crystal structure was then used for in silico screening of compounds, 11 of which were tested in vitro. Three compounds were found to be allosterically competitive with dopamine. One of the three activated TH moderately at high micromolar concentrations in the absence of dopamine, while the other two compounds markedly reduced activity in the absence of dopamine low micromolar range.
- Subject
- tyrosine hydroxylase; catecholamine; dopamine; salsolinol; enzyme kinetics; mutagenesis
- Identifier
- http://hdl.handle.net/1959.13/938514
- Identifier
- uon:12630
- Rights
- Copyright 2013 Gabrielle Briggs
- Language
- eng
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