1,721,039 research outputs found
Identification of the catalytic nucleophile of the family 31 alpha-glucosidase from aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate.
No full textThe mechanism-based reagent 5-fluoro-alpha-d-glucopyranosyl fluoride (5F alpha GlcF) was used to trap a glycosyl-enzyme intermediate and identify the catalytic nucleophile at the active site of Aspergillus niger alpha-glucosidase (Family 31). Incubation of the enzyme with 5F alpha GlcF, followed by peptic proteolysis and comparative liquid chromatography/MS mapping allowed the isolation of a labelled peptide. Fragmentation analysis of this peptide by tandem MS yielded the sequence WYDMSE, with the label located on the aspartic acid residue (D). Comparison with the known protein sequence identified the labelled amino acid as Asp-224 of the P2 subunit
Detailed dissection of a new mechanism for glycoside cleavage: ?-1,4-glucan lyase
No full textThe unusual enzyme, Gracilariopsis ?-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of ?-1,4-glucans via a ?-elimination reaction involving a covalent glycosyl?enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124, 4948?4949). The classical bell-shaped pH dependence of kcat/Km indicates two ionizable groups in the active site with apparent pKa values of 3.05 and 6.66. Brønsted relationships of log kcat versus pKa and log(kcat/Km) versus pKa for a series of aryl glucosides both show a linear monotonic dependence on leaving group pKa with low ?lg values of 0.32 and 0.33, respectively. The combination of these low ?lg values with large secondary deuterium kinetic isotope effects (kH/kD = 1.161.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (Ki = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-?-d-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large ?-secondary deuterium kinetic isotope effect (kH/kD = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (kH/kD = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining ?-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family
α-1,4-glucan lyase performs a trans-elimination via a nucleophilic displacement followed by a syn-elimination
No full text?-Glucan lyase (EC 4.2.2.13, GLase) cleaves ?-1,4-glucosidic bonds via an elimination reaction to produce 1,5-d-anhydrofructose. GLase was inactivated by the mechanism based ?-glucosidase inactivator, 5-fluoro-?-l-idosyl fluoride. The trapped glycosyl-enzyme intermediate was isolated and the nucleophilic amino acid residue (Asp 553) identified is equivalent to the residue so identified in sequence-related ?-glucosidases. This intermediate undergoes a syn-elimination reaction to release the product. Further evidence against a direct trans-elimination mechanism was provided by the absence of a primary kinetic isotope effect on the substrate substituted with deuterium at the C2 position
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Directed evolution of a glycosynthase from agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire
No full textThe Agrobacterium sp. ?-glucosidase (Abg) is a retaining ?-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ?-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ?-D-galactopyranosyl fluoride and 4-nitrophenyl ?-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6
Variations on the Author
Get PDF“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
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe 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
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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