STRUCTURAL STUDIES OF TYROSINE HYDROXYLASE IN THE APO-ENZYME AND INHIBITOR-BOUND STATES. Kenneth E.Goodwill*, Cara B. Marks, Christelle Sabatier, & Raymond C. Stevens, Chemistry Department, University of California, Berkeley 94720

Tyrosine hydroxylase (TH) is the rate limiting step in catecholamine (dopamine, adrenaline, and noradrenaline) biosynthesis. It is a member of the closely homologous family of aromatic amino acid hydroxylases. These hydroxylases require iron and biopterin as cofactors to add one atom from molecular oxygen to the aromatic ring. To date, no crystal structures have been solved in this family of enzymes.

The catalytic domain of rat TH has been cloned and expressed (Daubner, S.C. et al(1993). Protein Science 2, 1452-1460). This protein is as catalytically active as the whole enzyme. Both holo-TH and the catalytic domain have been shown to form a tetramer in solution.

Crystals of this enzyme have been grown from ammonium sulfate. Data for the apo-enzyme have been collected to a resolution of 2.3Å at SSRL beamline 7-1. The space group is F222 with unit cell dimensions of a=59.3Å, b=151.5Å, c=192.7Å. There is one monomer in the asymmetric unit.

A conformational change occurs when the crytals are soaked with 3-iodotyrosine, a clinically prescribed inhibitor of TH. The soaked crystals have two monomers in the asymmetric unit. The new space group is C222₁ with unit cell dimensions of a=72.6Å, b=154.1Å, c=155.5Å. Data have been collected from the inhibitor-bound form to 2.6Å resolution also at SSRL beamline 7-1.

Multiple isomorphous replacement is being used to determine the structures for both space groups (derivatives - Hg, Pb, Au, and seleno-methionine). Several good derivative data sets have been collected both with a rotating anode source and at SSRL.

Data is also being collected for a complex containing all participants in the reaction - iron, pterin and substrate or substrate analog. Additionally, a physiologically relevant complex containing the feed-back inhibitor dopamine is also being studied.

We would like to thank our collaborator in this project, Paul Fitzpatrick from Texas A&M University.