Tetrahydrobiopterin binding to aromatic amino acid hydroxylases. Ligand recognition and specificity.

The three aromatic amino acid hydroxylases (phenylalanine, tyrosine, and tryptophan hydroxylase) and nitric oxide synthase (NOS) all utilize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) as cofactor. The pterin binding site in the three hydroxylases is well conserved and different from the binding site in NOS. The structures of phenylalanine hydroxylase (PAH) and of NOS in complex with BH4 are still the only crystal structures available for the reduced cofactor−enzyme complexes. We have studied the enzyme-bound and free conformations of BH4 by NMR spectroscopy and molecular docking into the active site of the three hydroxylases, using endothelial NOS as a comparative probe. We have found that the dihydroxypropyl side chain of BH4 adopts different conformations depending on which hydroxylase it interacts with. All the bound conformations are different from that of BH4 free in solution at neutral pH. The different bound conformations appear to result from specific interactions with nonconserved amino aci...