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Isotope effect as a probe of the catalytic rilevance of coenzyme isomerization in the enzyme 6-phosphogluconate dehydrogenase from sheep liver
No full textCysteine 365 may have a role in the catalytic mechanism of 6-Phosphogluconate Dehydrogenase from Sheep Liver
No full text6-Phosphogluconate Dehydrogenase (6PGDH) is the third enzyme in the Pentose Phosphate Pathway and catalyzes the oxidative decarboxylation of 6-Phosphogluconate (6PG) to Ribulose 5-phosphate (Ru5P) with the concomitant reduction of NADP to NADPH. A general base/general acid mechanism has been suggested on the basis of pH dependence of kinetic parameters. Recent site-specific mutagenesis studies have indicated the Lysine 183 (K183) and the Glutammate 190 (E190) as best candidate for the role of catalytic base and acid, respectively. The crystal structure of the apoenzyme shows K183 protonated, whilst in the complex with the substrate it appear unprotonated. The missing proton is supposed to be accepted from a not well recognized residue which presumably may play a an relevant role in the catalytic mechanism of the enzyme. We believe that this process may be assisted by a Cysteine found abnormally reactive only in absence of substrate. We suggest that the most probable Cysteine, amongst the seven of 6PGDH, may be the C372 because it is the nearest to K183 and it is conserved in all sequences of the enzyme. Due to the proximity of Hystidine 186 to the two residues, and the high ionization reversibility of its side-chain, it may be hypothesized that the Hystidine could act as bridge in the exchange of the proton between K183 and C372.
We change C365 in a Serine,a Valine and a Aspartate in order to evaluate if the function of the residue is crucial in the catalysis of 6PGDH. Three mutations for the Cys 365 (Serine, Valine and Aspartate) were obtained by a in vitro site-specific mutagenesis system performed on the 6PGDH recombinant gene. All three resulting 6PGDH cDNA containing the mutation were cloned into a pQE-30 plasmid in the host strain M15[pREP4]. Each mutation was verified by sequence analysis. All mutant proteins were purified with a Ni-NTA resin, taking advantage of a six-His tag added to the N-terminus all 6PGDH derivatives. Neither CD nor fluorimetric analysis revealed evident difference in the gross structure of the mutant enzymes with respect to that of WT.Initial velocity pattern were performed spectrophotometically measuring the appearance of NADPH at 340 nm in the direction of oxidative decarboxylation. The pH dependence of kinetic parameters was assessed using three different buffer, in order to cover a pH range from 4.0 to 9.0. The primary deuterium isotope effects were determined fluorimetrically measuring the reduced coenzyme fluorescence at 460 nm, with excitation at 340 nm. The substitutions of C365 with Aspartate, Serine and Valine cause a decline in V/Et by at least 2 order of magnitude with respect to WT. On the contrary, the affinity constants, for both coenzyme (NADP) and substrate, do not show marked differences if compared to that of WT. Nevertheless, a small tendency in improving the affinity of 6PG has been observed (mostly in C365S where a KM three times lower has been found). The kinetic parameters of each mutant is pH-dependent as it occurs in WT, decreasing at low and high pH with slopes of 1 and –1, respectively. Thus, two independent pK, one in the acid side of the pH profile the other in the basic side, can be obtained by fitting the experimental data. The calculated values remarkably differ from those of WT (the pKb of the Serine mutant is around 1.0 pH) indicating that the substitution of 365C with the three amminoacids have perturbated the ionization constant of both catalytic residues of the enzyme. These data are consistent with a crucial role of C365 in modulating the pK of K183, which during the catalysis alternately acts as base and acid. The function of the residue may be that of shuttling the proton with the probable mediation of the proximal H183. The ability of this Hystidine to allow the transfer of the proton from inside to outside, and inversely, of catalytic system is going to be studied by further mutagenesis experiments
Proper orientation of the nicotinamide ring of NADP is important for the precatalytic conformational change in the 6-phosphogluconate dehydrogenase reaction
No full textA recent study suggested sheep liver 6-phosphogluconate dehydrogenase (6PGDH) sees the oxidized and reduced cofactor differently [Cervellati, C., Dallocchio, F., Bergamini, C. M., and Cook, P. F. (2005) Biochemistry 44, 2432-2440]. Data were consistent with a rotation into the active site of the nicotinamide ring of NADP upon its reduction, resulting in a displacement of the 1-carboxylate of 3-keto-6PG better positioning it for decarboxylation, and further suggested a role of the cofactor in generating the precatalytic conformation of the enzyme. To further probe the role of the cofactor, multiple isotope effects were measured for the enzyme with mutations of the two residues that directly interact with the nicotinamide ring of NADP+, methionine 13 and glutamate 131. Kinetic and isotope effect data obtained in this study will thus be interpreted in terms of a mechanism that includes the rotation of the nicotinamide ring. The M13V, M13Q, M13C, and E131A mutant enzymes were characterized with respect to their kinetic parameters, deuterium, 13C, multiple deuterium/13C isotope effects, and the kinetics of utilization of 2-deoxy-6PG. Data suggest the position of the nicotinamide ring is important in identifying the open and closed conformations of the enzyme, with the latter optimal for catalysis. The 6PGDH reaction provides an excellent example of the use of substrate binding energy to drive catalysi
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Possibile role of oxidative stress in the pathogenesis of chronic venous insufficiency. Preliminary results of a population study
No full textAppropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Influence of Menopausal Status and body fat distribution on oxidative stress” Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBIOC), Torino, September 19th-22nd 2006
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