Proton NMR Studies on the Conformation of the Pterin Cofactor Bound at the Active Site of Recombinant Human Tyrosine Hydroxylase

Abstract

The binding of L-erythro-7,S-dihydrobiopterin (BH₂) in the presence of L-Phe to recombinant human tyrosine hydroxylase was studied by ¹H-NMR spectroscopy. The distances (± 1.3 A) from the active site metal were estimated to be 5 .3-6.4 A for observable protons of BH₂ and 7 .0-7.9 A for L-Phe protons. Due to the lack of constraints from the pyrimidine ring in BH2 , two families of conformers were computed, with an average distance from the C4a in the pterin ring to the iron of 3.7 A (family A) and S.S A (family B). The conformers for 6-methyl-tetrahydropterin bound to the enzyme at near anaerobic conditions in the absence of L-Phe were also grouped into two families, with C4a-metal distances of 3.0-4.0 A (A) and 7.0-7.5 A (B). Based on the estimated conformation of the enzyme bound 5,6,7,S-tetrahydro- 3-methylpterin, family A of conformers was selected for both oxidized and reduced pterins. Our results indicate that the pterin does not coordinate to the iron, which seems to be involved both in the binding and activation of O₂ and in the C-O bond formation in the aromatic ring of the substrate.