CRYSTAL-STRUCTURE OF TYROSINE-HYDROXYLASE WITH BOUND COFACTOR ANALOG AND IRON AT 2.3 ANGSTROM RESOLUTION - SELF-HYDROXYLATION OF PHE300 ANDTHE PTERIN-BINDING SITE

TyrOH is a non-heme iron enzyme which uses molecular oxygen to hydroxy late tyrosine to form L-dihydroxyphenylalanine (L-DOPA), and tetrahydr obiopterin to form 4a-hydroxybiopterin in the rate-limiting step of th e catecholamine biosynthetic pathway. The 2.3 Angstrom crystal structu re of the catalytic and tetramerization domains of rat tyrosine hydrox ylase (TyrOH) in the presence of the cofactor analogue 7,8-dihydrobiop terin and iron shows the mode of pterin binding and the proximity of i ts hydroxylated 4a carbon to the required iron. The pterin binds on on e face of the large active-site cleft, forming an aromatic pi-stacking interaction with Phe300. This phenylalanine residue of TyrOH is found to be hydroxylated in the meta position, most likely through an autoc atalytic process, and to consequently form a hydrogen bond to the main -chain carbonyl of Gln310 which anchors Phe300 in the active site. The bound pterin forms hydrogen bonds from N-8 to the main-chain carbonyl of Leu295, from O-4 to Tyr371 and Glu376, from the C-1' OH to the mai n-chain amides of Leu294 and Leu295, and from the C-2' hydroxyl to an iron-coordinating water. The part of the pterin closest to the iron is the O-4 carbonyl oxygen at a distance of 3.6 Angstrom. The iron is 5. 6 Angstrom from the pterin 4a carbon which is hydroxylated in the enzy matic reaction. No structural changes are observed between the pterin bound and the nonliganded enzyme. On the basis of these structures, mo lecular oxygen could bind in a bridging position optimally between the pterin C-4a and iron atom prior to substrate hydroxylation. This stru cture represents the first report of close interactions between pterin and iron in an enzyme active site.