I. Name, Wesen und Abgrenzung des Begriffs
Der Begriff Kirchenlied bezeichnet »einen … geistlichen Text christlicher Prägung, gleichgültig welchen Bekenntnisses, in metrischer Form von stroph...
Tryptophan hydroxylase (TPH) carries out the 5-hydroxylation of l-Trp, which is the rate-limiting step in the synthesis of serotonin. We have prepared and characterized a stable N-terminally ...truncated form of human TPH that includes the catalytic domain (Δ90TPH). We have also determined the conformation and distances to the catalytic non-heme iron of both l-Trp and the tetrahydrobiopterin cofactor analogue l-erythro-7,8-dihydrobiopterin (BH2) bound to Δ90TPH by using 1H NMR spectroscopy. The bound conformers of the substrate and the pterin were then docked into the modeled three-dimensional structure of TPH. The resulting ternary TPH·BH2·l-Trp structure is very similar to that previously determined by the same methods for the complex of phenylalanine hydroxylase (PAH) with BH2 and l-Phe Teigen, K., et al. (1999) J. Mol. Biol. 294, 807−823. In the model, l-Trp binds to the enzyme through interactions with Arg257, Ser336, His272, Phe318, and Phe313, and the ring of BH2 interacts mainly with Phe241 and Glu273. The distances between the hydroxylation sites at C5 in l-Trp and C4a in the pterin, i.e., 6.1 ± 0.4 Å, and from each of these sites to the iron, i.e., 4.1 ± 0.3 and 4.4 ± 0.3 Å, respectively, are also in agreement with the formation of a transient iron-4a-peroxytetrahydropterin in the reaction, as proposed for the other hydroxylases. The different conformation of the dihydroxypropyl chain of BH2 in PAH and TPH seems to be related to the presence of nonconserved residues, i.e., Tyr235 and Pro238 in TPH, at the cofactor binding site. Moreover, Phe313, which seems to interact with the substrate through ring stacking, corresponds to a Trp residue in both tyrosine hydroxylase and PAH (Trp326) and appears to be an important residue for influencing the substrate specificity in this family of enzymes. We show that the W326F mutation in PAH increases the relative preference for l-Trp as the substrate, while the F313W mutation in TPH increases the preference for l-Phe, possibly by a conserved active site volume effect.
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 acids at the BH4 binding sites of the hydroxylases, notably the stretch 248−251 (numeration in PAH) and the residue corresponding to Ala322 in PAH, i.e., Ser in TH and Ala in TPH. On the basis of analysis of molecular interaction fields, we discuss the selectivity determinants for each hydroxylase and explain the high-affinity inhibitory effect of 7-tetrahydrobiopterin specifically for PAH.
Congenital tracheo-oesophageal fistula without atresia can persist and remain undetected until adulthood. We present such a case and give a review of the literature. Symptoms are commonly present ...during infancy, but to confirm diagnosis of a fistula is often exceedingly difficult. We visualized the fistula in our case by means of a bronchofibrescope when all the other available tests had failed. The speculations as to how these patients reach adult life have been many, but we consider that we have found an important and hitherto undescribed answer to this question, namely a sphincter mechanism in the fistula.
