A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase

Tetrahydropterins are obligatory cofactors for tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. A series of synthetic analogues of 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) with different substituents in positions C2, N3, C4, N5, C6, C7, and N8 on the ring were used as active site probes for recombinant human TH. The enzyme tolerates rather bulky substituents at C6, as seen by the catalytic efficiency (V-max/K-m) and the couplings efficiency (mol of L-DOPA produced/mol of tetrahydropterin oxidized) of the cofactors. Substitutions at C2, C4, N5, and Ns abolish the cofactor activity of the pterin analogues. Molecular docking of BH4 into the crystal structure of the catalytic domain of ligand-free rat TH results in complexes in which the pteridine ring pi -stacks with Phe300 and the N3 and the amino group at C2 hydrogen bonds with Glu332. The pteridine ring also establishes interactions with Leu294 and Gln310. The distance between C4a in the pteridines and the active site iron was 4.2 +/- 0.5 Angstrom for the ensemble of docked conformers. Docking of BH4 analogues into TH also shows that the most bulky substituents at C6 can be well-accommodated within the large hydrophobic pocket surrounded by Ala297, Ser368, Tyr371, and Trp372, without altering the positioning of the ring. The pterin ring of 7-BH4 shows proper stacking with Phe300, but the distance between the C4a and the active site iron is 0.6 Angstrom longer than for bound BH4, a finding that may be related to the high degree of uncoupling observed for 7-BH4.