A Kinetic and Conformational Study on the Interaction of Tetrahydropteridines with Tyrosine Hydroxylase

Abstract

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 (BH(4)) 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 coupling efficiency (mol of L-DOPA produced/mol of tetrahydropterin oxidized) of the cofactors. Substitutions at C2, C4, N5, and N8 abolish the cofactor activity of the pterin analogues. Molecular docking of BH(4) 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 A for the ensemble of docked conformers. Docking of BH(4) 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-BH(4) shows proper stacking with Phe300, but the distance between the C4a and the active site iron is 0.6 A longer than for bound BH(4), a finding that may be related to the high degree of uncoupling observed for 7-BH(4).

Modeled ligand‐protein complexes elucidate the origin of substrate specificity and provide insight into catalytic mechanisms of phenylalanine hydroxylase and tyrosine hydroxylase

“…In this study, the distance between C4a of the pterin analog and the iron atom has been measured as 3.7 Å . Our coordinates of the pterin position are also in agreement with a recent study using the program DOCK 4.0 [37] to model the conformation of BH 4 and a series of pterin analogs placed into the crystal structure of the TH catalytic domain [15]. On the other hand, equivalent energy scores were obtained when BH 4 was manually docked into TH according to the orientation determined by X-ray crystallography [13].…”

“…3). This conformation of BH 4 coincided with the conformation of BH 4 in TH previously computed by Alma˚s et al using DOCK 4.0 (rmsd 2.2 ± 0.2 Å ) [15]. However, it differed from the conformation of 7,8-BH 2 in the binary complex identified by X-ray crystallography [13].…”

“…X-ray crystallography of 7,8-dihydrobiopterin (7, bound to truncated rat TH and human PAH has identified amino acid residues critical for the positioning of this oxidized cosubstrate in the second coordination sphere of the catalytic iron [13,14]. The impact of the structural identity of the pterin cosubstrate on TH activity has been shown in a kinetic study using synthetic pterin analogs [15]. Spectroscopic investigations of TH and crystallographic data obtained from binary complexes of catecholamines and truncated human PAH have demonstrated that the two hydroxyl groups of the catechol moiety bind to the catalytic iron [16][17][18].…”

Peroxynitrite-induced Nitration of Tyrosine Hydroxylase

“…This is not that surprising when considering that this hydrophobic pocket accommodates numerous bulky substitutions at the C-6 position of the pterin cofactor (38,39). Tyr 423 and Tyr 428 lie within one of two loops, the other of which is composed of residues 290 -296, that reach over the active site opening of the TH (37,38).…”

“…Ϫ pterin cofactor binding (35,38,39), yet it tolerates mutagenesis to phenylalanine very well. This is not that surprising when considering that this hydrophobic pocket accommodates numerous bulky substitutions at the C-6 position of the pterin cofactor (38,39).…”