The Conformation of Tetrahydro-Biopterin Free and Bound to Aromatic Amino Acid Hydroxylases and NOS

Abstract

Phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH) and nitric oxide synthase (NOS) are tetrahydrobiopterin (BH₄)-dependent enzymes that catalyze the hydroxylation of the respective aromatic amino acids (PAH, TH and TPH) and the synthesis of NO from arginine (NOS), using dioxygen as additional substrate. While the aromatic amino acid hydroxylases all contain a catalytic mononuclear non-heme iron which is essential for the hydroxylation, NOS contains a cytochrome P450-type heme in the oxygenase domain where NO synthesis seems to take place. We have recently studied the structure of the complex of BH₂ (the oxidized analogue of BH₄) and substrate with PAH by NMR and docking 1 and, in order to get further insights on the role of the iron and tetrahydropterin cofactor in the catalytic mechanism in these enzymes, we have extended these studies to BH₄. Based on the distance constraints obtained by NMR complemented by distance geometry calculations, docking into the crystal structure of the enzymes and molecular dynamic simulations, we have determined the conformation of BH₄ bound to each of the four enzymes.