scholarly journals Identification of Glu-519 as the catalytic nucleophile in β-mannosidase 2A from Cellulomonas fimi

2000 ◽  
Vol 351 (3) ◽  
pp. 833-838 ◽  
Author(s):  
Dominik STOLL ◽  
Shouming HE ◽  
Stephen G. WITHERS ◽  
R. Antony J. WARREN
Keyword(s):  
Peptide Bond ◽  
Enzyme Inactivation ◽  
Ionization Mass ◽  
Glycosyl Hydrolases ◽  
Cellulomonas Fimi ◽  
Inactivation Rate Constant ◽  
Dependent Inactivation ◽  
The Difference ◽  
The Masses ◽  
Covalent Intermediate

Incubation of the β-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-β-d-mannosyl fluoride (2FManβF) resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki = 0.57min-1, dissociation constant for the inactivator Ki = 0.41mM) through the accumulation of a covalent 2-deoxy-2-fluoro-α-d-mannosyl–β-mannosidase 2A (2FMan–Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry. The stoichiometry of inactivation was 1:1. Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose [Glcβ(1–6)Glc] demonstrated that the covalent intermediate was catalytically competent. Comparison by MS of the peptic digests of 2FMan–Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan–Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide. Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517–528 and 520–528 of Man2A respectively. The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose. It was concluded that in 2FMan–Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519–Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme. This residue is conserved in all members of family 2 of the glycosyl hydrolases. This represents the first ever labelling and identification of an active-site nucleophile in a β-mannosidase.

scholarly journals Identification of Glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum β-xylosidase using electrospray MS

1998 ◽  
Vol 335 (2) ◽  
pp. 449-455 ◽  
Author(s):  
David J. VOCADLO ◽  
Lloyd F. MACKENZIE ◽  
Shouming HE ◽  
Gregory J. ZEIKUS ◽  
Stephen G. WITHERS
Keyword(s):  
Neutral Loss ◽  
Enzyme Inactivation ◽  
Peptide Sequence ◽  
Glycosyl Hydrolases ◽  
Inactivation Rate Constant ◽  
Thermoanaerobacterium Saccharolyticum ◽  
Dependent Inactivation ◽  
Peptic Digestion ◽  
Covalent Intermediate ◽  
Enzyme Intermediate

Thermoanaerobacterium saccharolyticum β-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining β-d-xylosidases and α-l-iduronidases. T. saccharolyticum β-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl–enzyme intermediate. Incubation of T. saccharolyticum β-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki = 0.089 min-1, dissociation constant for the inactivator Ki = 65 µM) through the accumulation of a covalent 2-deoxy-2-fluoro-α-d-xylosyl–enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-α-d-xylosyl–enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-α-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.

A study of the relationships of interactions between Asp-201, Na+ or K+, and galactosyl C6 hydroxyl and their effects on binding and reactivity of β-galactosidase

2004 ◽  
Vol 82 (2) ◽  
pp. 275-284 ◽  
Author(s):  
Julia Xu ◽  
Mary A.A McRae ◽  
Scott Harron ◽  
Beatrice Rob ◽  
Reuben E Huber
Keyword(s):  
Physical Properties ◽  
Negative Charge ◽  
Binding Pocket ◽  
Side Chain ◽  
Wild Type ◽  
Sodium Potassium ◽  
The Difference ◽  
Potassium Binding ◽  
Covalent Intermediate ◽  
Catalytic Effects

The interactions between Na+ (and K+) and Asp-201 of β-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type β-galactosidase (0.36 ± 0.09 mM) was about 10× lower than for K+ (3.9 ± 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-β-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-β-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.Key words: β-galactosidase, sodium, potassium, binding, aspartic acid.

scholarly journals Study of the Abundance Patterns in the Metal-Poor Stellar Stream

2013 ◽  
Vol 30 ◽  
Author(s):  
Hongjie Li ◽  
Shuai Liang ◽  
Wenyuan Cui ◽  
Bo Zhang
Keyword(s):  
Gradual Increase ◽  
Type Ii ◽  
Constant Mass ◽  
Chemical Abundances ◽  
Abundance Patterns ◽  
The Difference ◽  
The Masses ◽  
Process Component ◽  
Process Elements ◽  
Early Galaxy

AbstractThe chemical abundances of the metal-poor stars in the stellar stream provide important information for setting constraints on models of neutron-capture processes. The study of these stars could gives us a better understanding of the r-process nucleosynthesis and chemical composition of the early Galaxy. Using the updated main r-process and weak r-process patterns, we fit abundances in the stellar stream stars. The weak r-process component coefficients are almost constant for the sample stars, including r-rich stars, which means that both the weak r-process and Fe are produced as primary elements from Type II supernovae and their yields have nearly a constant mass fraction. The difference between the stream stars and r-rich stars is obvious. For the stream stars, the fact that the increased trend in the main r-process component coefficients as metallicity increases means a gradual increase in the production of main r-process elements relative to iron. This behaviour implies that the masses of progenitors for the main r-process are smaller than those of the weak r-process. Furthermore, we find that the metal-poor stream star HD 237846 is a weak r-process star.

Mannan-Degrading Enzymes fromCellulomonas fimi

1999 ◽  
Vol 65 (6) ◽  
pp. 2598-2605 ◽  
Author(s):  
Dominik Stoll ◽  
Henrik Stålbrand ◽  
R. Antony J. Warren
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unknown Function ◽  
Glycosyl Hydrolases ◽  
Intracellular Enzyme ◽  
Cellulomonas Fimi ◽  
Catalytic Module ◽  
Mannanase Activity ◽  
Homology Module ◽  
Active Fragments

ABSTRACT The genes man26a and man2A fromCellulomonas fimi encode mannanase 26A (Man26A) and β-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced byEscherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian β-mannosidases.

scholarly journals Fundamental properties of Kepler and CoRoT targets – IV. Masses and radii from frequencies of minimum Δν and their implications

2019 ◽  
Vol 489 (2) ◽  
pp. 1753-1769 ◽  
Author(s):  
M Yıldız ◽  
Z Çelik Orhan ◽  
C Kayhan
Keyword(s):  
Rotational Velocity ◽  
Galactic Disc ◽  
Ionization Zone ◽  
Chemical Enrichment ◽  
Fundamental Properties ◽  
Diagnostic Potential ◽  
The Difference ◽  
The Masses ◽  
Oscillation Frequencies

ABSTRACT Recently, by analysing the oscillation frequencies of 90 stars, Yıldız, Çelik Orhan & Kayhan have shown that the reference frequencies (νmin0, νmin1, and νmin2) derived from glitches due to He ii ionization zone have very strong diagnostic potential for the determination of their effective temperatures. In this study, we continue to analyse the same stars and compute their mass, radius, and age from different scaling relations including relations based on νmin0, νmin1, and νmin2. For most of the stars, the masses computed using νmin0 and νmin1 are very close to each other. For 38 stars, the difference between these masses is less than 0.024 M$\odot$. The radii of these stars from νmin0 and νmin1 are even closer, with differences of less than 0.007 R$\odot$. These stars may be the most well known solar-like oscillating stars and deserve to be studied in detail. The asteroseismic expressions we derive for mass and radius show slight dependence on metallicity. We therefore develop a new method for computing initial metallicity from this surface metallicity by taking into account the effect of microscopic diffusion. The time dependence of initial metallicity shows some very interesting features that may be important for our understanding of chemical enrichment of Galactic Disc. According to our findings, every epoch of the disc has its own lowest and highest values for metallicity. It seems that rotational velocity is inversely proportional to 1/2 power of age as given by the Skumanich relation.

scholarly journals Some experiments on thermal diffusion

1921 ◽  
Vol 99 (700) ◽  
pp. 385-397 ◽  
Keyword(s):  
Steady State ◽  
Temperature Gradient ◽  
Thermal Diffusion ◽  
General Effect ◽  
Gas Molecule ◽  
Calculated Effect ◽  
Order Of Magnitude ◽  
Gas Molecules ◽  
The Difference ◽  
The Masses

It has been shown theoretically by Dr. S. Chapman that a temperature gradient applied to a uniform mixture of two gases will tend to produce non-uniformity of composition, the heavier and larger molecules diffusing towards the cooler side, and the smaller and lighter molecules diffusing towards the hotter side. This phenomenon was termed “thermal diffusion.” The difference in composition due to thermal diffusion increases until it is balanced by the opposite effects of ordinary diffusion, when a steady state will be reached. The effect will be greatest when the gases are mixed in nearly equal proportions by volume, and will be greater the more unequal are the masses and diameters of the gas molecules. It was also shown that the extent of the effect would vary with the character of the gas molecules, being at a maximum when the molecules behave like rigid elastic spheres. In the case where molecules behave like fifth power centres of force the effect would disappear entirely. Experiments made by Chapman and Dootson established the existence of the phenomenon. The results were chiefly qualitative, the nature and order of magnitude being in agreement with the theory. In no case were the differences equal to the theoretical values obtained on the assumption that gas molecules behave like rigid elastic spheres. Although the results are not claimed to be exact, the general effect could be regarded as rather less than a half of this calculated effect. The character of the gas molecule required to give this result would, however, be in agreement with that obtained for the actual gas molecules from the investigation of the variation of viscosity with temperature.

scholarly journals Maturation of IncP Pilin Precursors Resembles the Catalytic Dyad-Like Mechanism of Leader Peptidases

2000 ◽  
Vol 182 (23) ◽  
pp. 6751-6761 ◽  
Author(s):  
Ralf Eisenbrandt ◽  
Markus Kalkum ◽  
Rudi Lurz ◽  
Erich Lanka
Keyword(s):  
Amino Acid ◽  
Peptide Bond ◽  
Ring Structure ◽  
Site Directed Mutagenesis ◽  
Signal Peptidase ◽  
Ionization Mass ◽  
Preparation Technique ◽  
Core Sequence ◽  
Specific Phage ◽  
N Terminus

ABSTRACT The pilus subunit, the pilin, of conjugative IncP pili is encoded by the trbC gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M. Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548–22555, 1999). Three enzymes are involved in maturation of the pilin: LepB ofEscherichia coli for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide. We refer to the latter process as “prepilin cyclization.” We have used site-directed mutagenesis of trbC and traF to unravel the pilin maturation process. Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities. Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization–mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.

scholarly journals A new specimen-dependent method of estimating felid body mass

2016 ◽  
Author(s):  
Shaheer Sherani
Keyword(s):  
Body Mass ◽  
The Body ◽  
Long Bone ◽  
Panthera Tigris ◽  
Living Species ◽  
Smilodon Fatalis ◽  
The Difference ◽  
The Masses ◽  
Bone Dimensions ◽  
Panthera Atrox

Background. The estimation of body mass of long extinct species of the family Felidae has been a focus of paleontology. However, most utilized methods impose expected proportions on the fossil specimens being estimated, resulting in a high chance of underestimation or overestimation. This study proposes a new method of estimating felid body mass by accounting for osteological proportionality differences between the extinct taxa being estimated and the living species being used as comparisons. Method. Using a manipulation of the cube law, 36 equations were formulated that estimate body mass based on certain humeral and femoral dimensions. The formulated equations were used to examine whether the mass of living comparison species, namely the tiger (Panthera tigris), the lion (Panthera leo), and the jaguar (Panthera onca), depends equally on a select set of long bone dimensions. The body mass of five extinct felids, namely Panthera atrox, Panthera spelaea, Panthera tigris soloensis, Smilodon populator, and Smilodon fatalis, was also estimated. Results. Living comparisons species were found to somewhat incorrectly estimate other living comparison species. All five extinct taxa were found to weigh well over 300 kg, with the largest of the species weighing nearly 500 kg. Discussion. The inability of one living comparison species to predict the mass of another with strong accuracy suggests that bone dimensions do not solely influence body mass. Discrepancies between the masses of Smilodon populator and Smilodon fatalis were likely the product of the difference in available niches in late Pleistocene North and South America. The masses of Panthera spelaea and Panthera atrox indicate a discrepancy in sociality between the two closely related species. Lastly, the extreme body mass of Panthera tigris soloensis points to great plasticity within the tiger lineage in terms of size, indicating that such variations among tiger populations may not warrant subspeciation.

scholarly journals Historical and Evolutionary Analysis of Reasons and Causes of Objections of Orientalism on Prophet’s Biography

2020 ◽  
Vol 2 (1) ◽  
pp. 274-296
Author(s):  
Bakht Shaid ◽  
Hafiz Muhammad Sani
Keyword(s):  
Large Scale ◽  
Human Society ◽  
Evolutionary Analysis ◽  
Not Given ◽  
Islamic History ◽  
Difference Of Opinion ◽  
The Difference ◽  
Mutual Relations ◽  
The Masses ◽  
Islamic Civilization

سیرت طیبہ پر استشراقی فکر کے اعتراضات کے اسباب و عوامل کا تاریخی و ارتقائی جائزہ Over the course of time and with the rapid increase in human population need for mutual relations become crucial. Resultantly on behalf of this closeness, separation, anti-standpoints and comparisons also emerged. As the time passed by hatred and hypocrisy and other social vices spread on large scale. Thus human society was waiting for such liberator who may lead and work for the betterment of this society. With the dawn of Islamic civilization all such issues were not only resolved but also provided with a model for containing the difference of opinion and multiple traditions under its unique worldview. Islamic History presents itself as a model where the minorities were provided with the opportunities of participating in political, social, educational and collective affairs. Thus in a society where tyranny, injustice, un-forbearance, religious intensity, terrorism and the activities of violating the human rights were very common, were substituted by the Islamic ideal  of forbearance. It is argued here that the solution of all these issues was only in religion contrary to what is being claimed about an idea of social harmony where religion is not given its due position. Today its our dire need to develop a sense of harmony, modesty, affection and peacefulness among the masses of various religions of Pakistani society. It is further argued that for this very noble cause all the religious scholars and their followers can come forward playing their pertinent role.  

scholarly journals ON THE QUESTION OF MODERN MYTHS. POLITICAL MYTH AS APPLIED MYTHOLOGY

2021 ◽  
Vol 17 (1) ◽  
pp. 44-50
Author(s):  
Victoria Omelchenko
Keyword(s):  
The Political ◽  
Dominant Position ◽  
World Picture ◽  
Scientific Exploration ◽  
Political Myth ◽  
Political Speeches ◽  
The Difference ◽  
Philosophical Texts ◽  
Political Mythology ◽  
The Masses

In this article, the author argues that philosophy is a "methodology" of understanding. Hermeneutics is the theory and methodology of interpretation especially the interpretation philosophical texts. H.-G. Gadamer asserted that methodical contemplation is opposite to experience and reflection. We can reach the truth only by understanding or mastering our experience. According to H.-G. Gadamer, our understanding is not fixed but rather is changing and always indicating new perspectives. The most important thing is to unfold the nature of individual understanding. Hermeneutics is not just "art", but the methodology of "understanding." Accordingly, philosophy is the methodology of understanding and interpretation. Philosophy is a science with its own subject and object. Legal laws are not absolute, therefore they should be interpreted. It is the task of philosophy. Philosophy speaks the language of concepts and evidence. Concepts should be specific, clear, defined. Philosophical work is the formulation of concepts. On the basis of such understanding of philosophy, we will now study the question of "political myth". Scientific exploration is devoted to the study of the concept of "myth" and identifying the essential features of the "political myth". What is "political myth" of modernity? What language is he speaking? What appealed? In this intelligence proved that "political myth" is a communication system that distorts reality. It is established that every "political myth" has its own shelf life and the boundaries of its interpretation. It is confirmed that "political myth" is one of the most effective tools to meet political goals, such as achievement of power and its legitimation. It is proved that political discourse is irrational, and political speeches appeal to the argument of sensuous, not rational. "Political myth" models own "world picture", which is a simplified, taken for granted, self-evident. It is proved that the ability of critical judgment, ability to work with information and qualitative analysis from the mouth of the unbiased media and the expert environment, though not a panacea, but partially negate the "force," "political myth". Myth has an irrational basis, and controls the emotions with the help of stereotypes. The myth creates its own reality and creates the effect of solidarity of the masses. The essence of "political myth" is that he always addressed to specific audiences, takes into account the peculiarities of mentality. A dominant position in the "political myth" plays a stereotype. Any stereotype is formed on the generalizations. What is the difference between the stereotypes and myths? "A stereotype is a label", which is the context, but it there is no story. That is, the stereotype is a form, and the myth is content history. In addition, in this exploration of the role and value of such concepts as "political magic" and "political spectacle" in the context of "political mythology" as applied mythology.

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