Conformation of the substrate and pterin cofactor bound to human tryptophan hydroxylase. Important role of Phe313 in substrate specificity.

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 (Delta90TPH). 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 Delta90TPH 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 A, and from each of these sites to the iron, i.e., 4.1 +/- 0.3 and 4.4 +/- 0.3 A, 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.

Why is p = .90 better than p = .70? Preference for definitive predictions by lay consumers of probability judgements.

What do people regard as an informative and valuable probability statement? This article reports four experiments that show participants to have a clear preference for more extreme and higher probabilities over less extreme and lower ones. This pattern emerged in Experiment 1, in which no context was provided, and was further explored in Experiment 2 within a positive and a negative context. The findings were further confirmed in Experiment 3, which employed a Bayesian framework with revisions of opinions. Finally, Experiment 4 showed how preference for high probabilities can lead people to prefer an overconfident to a more well-calibrated (accurate) forecaster. The results are interpreted as manifestations of a search for definitive predictions principle, which asserts that high probabilities are preferred to medium ones and often favored over the corresponding complementary low probabilities on the basis of their capacity to predict the occurrence of single outcomes.

The probability-outcome correspondence principle: a dispositional view of the interpretation of probability statements.

This article presents a framework for lay people's internal representations of probabilities, which supposedly reflect the strength of underlying dispositions, or propensities, associated with the predicted event. From this framework, we derive the probability-outcome correspondence principle, which asserts that strong dispositions should lead to (1) strong (forceful) and (2) immediate outcomes and, hence, be characterized by high probabilities. In contrast, weak dispositions lead to (1) weak (fragile) and (2) delayed outcomes and are thus associated with low probabilities. We describe six experiments designed to test the correspondence principle. In the final discussion, we examine the implications of the proposed framework, from both a normative and a descriptive viewpoint.

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 (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).

The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism.

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.

Hazards mean luck: counterfactual thinking in reports of dangerous situations and careless behavior.

It has been shown (Teigen, 1995) that experiences of "luck" in daily life are dependent upon the existence a worse and close hypothetical (counterfactual) outcome, rather than upon a positive evaluation of what actually happened. The present investigation focuses on the inverse relationship, namely whether a situation with a negative outcome close at hand will be perceived as lucky. To test this hypothesis, students were asked to describe dangerous situations (Experiment 1) and examples of careless behavior (Experiment 3) from their own lives, which subsequently were rated by the actors and by peer groups for good and bad luck, attractiveness, and for closeness and attractiveness of the counterfactual outcome. Dangerous situations and episodes involving careless behavior were generally regarded as more lucky than unlucky. Furthermore, degree of good luck was positively correlated with degree of dangerousness and with degree of carelessness. Luck was related to closeness, aversiveness, and (in Experiment 2) to estimated probability of the counterfactual outcome. It is concluded that luck is primarily determined by negative outcomes that did not happen, and thus a frequent by-product of risk taking and risk exposure.

In vitro activity of 9 antimicrobial agents against 177 strains of Haemophilus influenzae isolated from hospitalized patients.

In vitro activity of 4 commonly used and 5 new antibiotics was examined against 177 strains of Haemophilus influenzae. All strains were collected from various sites in patients with clinical infections. The study confirms that several newer antibiotics are useful alternatives to older drugs, as measured by in vitro activity. Ciprofloxacin and ofloxacin were the most active agents, (MIC90 0.012 micrograms/ml and 0.05 micrograms/ml respectively), followed by aztreonam (MIC90 0.1 micrograms/ml) and cefuroxime (MIC90 0.8 micrograms/ml). A new macrolide, azithromycin (CP 62,993), was more active than erythromycin, MIC90 1.6 micrograms/ml vs 6.4 micrograms/ml. Beta-lactamase production was detected in 4.5% (8/177) of the strains. In vitro activity was the same against strains collected in 1985 and 1988. No increase in beta-lactamase production was recorded.

[Toxic shock syndrome without any relation to the use of tampons or staphylococcal infection]. / Toksisk sjokk-syndrom. Uten relasjon til tampongbruk eller stafylokokkinfeksjon.

Toxic shock syndrome is a generalized disease traditionally thought to be caused by toxinproducing strains of Staphylococcus aurus. The syndrome is characterized by fever, hypotension, erythema of the skin, erythematous desquamation and multiple organ involvement. We present a case where the causative agent was most probably streptococci, thus demonstrating that the syndrome probably has a heterogeneous, toxin-related etiology.