scholarly journals Proton release on binding of glutathione to Alpha, Mu and Delta class glutathione transferases

1999 ◽  
Vol 344 (2) ◽  
pp. 419-425 ◽  
Author(s):  
Anna Maria CACCURI ◽  
Giovanni ANTONINI ◽  
Philip G. BOARD ◽  
Michael W. PARKER ◽  
Maria NICOTRA ◽  
...  
Keyword(s):  
Flow Analysis ◽  
Three Dimensional ◽  
Kinetic Mechanism ◽  
Activation Mechanism ◽  
Dimensional Structure ◽  
Binary Complex ◽  
Stopped Flow ◽  
Binding Mechanism ◽  
Proton Extrusion ◽  
Evolutionary Pathway

Potentiometric, spectroscopic and stopped-flow experiments have been performed to dissect the binding mechanism of GSH to selected glutathione S-transferases (GSTs), A1-1, M2-2 and Lucilia cuprina GST, belonging to Alpha, Mu and Delta classes respectively. Both Alpha and Mu isoenzymes quantitatively release the thiol proton of the substrate when the binary complex is formed. Proton extrusion, quenching of intrinsic fluorescence and thiolate formation, diagnostic of different steps along the binding pathway, have been monitored by stopped-flow analysis. Kinetic data are consistent with a multi-step binding mechanism: the substrate is initially bound to form an un-ionized pre-complex [k1⩾ (2-5)×106 M-1˙s-1], which is slowly converted into the final Michaelis complex (k2 = 1100-1200 s-1). Ionization of GSH, fluorescence quenching and proton extrusion are fast events that occur either synchronously or rapidly after the final complex formation. The Delta isoenzyme shows an interesting difference: proton extrusion is almost stoichiometric with thiolate formed at the active site only up to pH 7.0. Above this pH, at least one protein residue acts as internal base to neutralize the thiol proton. These results suggest that the Alpha and Mu enzymes retain not only a similar catalytic outcome and overall three-dimensional structure but also share a similar kinetic mechanism for GSH binding. The Delta GST, which is closely related to the mammalian Theta class enzymes and is distantly related to Alpha and Mu GSTs in the evolutionary pathway, might display a different activation mechanism for GSH.

scholarly journals Three-Dimensional Structure of PANI/CdS NRs-SiO2 Hydrogel for Photocatalytic Hydrogen Evolution with High Activity and Stability

Nanomaterials ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 427 ◽  
Author(s):  
Jinrong Lu ◽  
Xin Zhang ◽  
Huiyuan Gao ◽  
Wenquan Cui
Keyword(s):  
Hydrogen Production ◽  
Active Sites ◽  
Production Efficiency ◽  
Conductive Polymer ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Composite Catalyst ◽  
Binary Complex ◽  
Three Dimensional Structure ◽  
Cds Nanorods

Three-dimensional PANI/CdSNRs-SiO2 hydrogel (CdS NRs-PANI-SiO2) was synthesized by loading polyaniline (PANI) onto the semiconductor CdS nanorods (NRs) surface and loading the binary complex on SiO2 gel. The structure, optical properties, and electrochemical properties of the composite were studied in detail. The hydrogen production amount of CdS NRs-PANI (3%)-SiO2 (20%) increased in comparison with CdS NRs and reached 43.25 mmol/g in 3 h under visible light. The three-dimensional structure of SiO2 hydrogel increased the specific surface area of the catalyst, which was conducive to exposing more active sites of the catalyst. In addition, the conductive polymer PANI coated on CdS NRs played the role of conductive charge and effectively inhibited the photo-corrosion of CdS NRs. In addition, the recovery experiment showed that the recovery rate of the composite catalyst reached 90% and hydrogen production efficiency remained unchanged after five cycles, indicating that the composite catalyst had excellent stability.

scholarly journals Thermophoretic analysis of ligand-specific conformational states of the inhibitory glycine receptor embedded in copolymer nanodiscs

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Max Bernhard ◽  
Bodo Laube
Keyword(s):  
Ligand Binding ◽  
Partial Agonist ◽  
Glycine Receptor ◽  
Three Dimensional ◽  
Receptor Activation ◽  
Cell System ◽  
Activation Mechanism ◽  
Hek293 Cells ◽  
Dimensional Structure ◽  
Conformational States

Abstract The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel family (pLGIC), displays remarkable variations in the affinity and efficacy of the full agonist glycine and the partial agonist taurine depending on the cell system used. Despite detailed insights in the GlyR three-dimensional structure and activation mechanism, little is known about conformational rearrangements induced by these agonists. Here, we characterized the conformational states of the α1 GlyR upon binding of glycine and taurine by microscale thermophoresis expressed in HEK293 cells and Xenopus oocytes after solubilization in amphipathic styrene-maleic acid copolymer nanodiscs. Our results show that glycine and taurine induce different conformational transitions of the GlyR upon ligand binding. In contrast, the variability of agonist affinity is not mediated by an altered conformational change. Thus, our data shed light on specific agonist induced conformational features and mechanisms of pLGIC upon ligand binding determining receptor activation in native environments.

scholarly journals Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

2007 ◽  
Vol 282 (46) ◽  
pp. 33572-33582 ◽  
Author(s):  
Judit Tóth ◽  
Balázs Varga ◽  
Mihály Kovács ◽  
András Málnási-Csizmadia ◽  
Beáta G. Vértessy
Keyword(s):  
Steady State ◽  
Active Site ◽  
Reaction Pathway ◽  
Three Dimensional ◽  
Kinetic Mechanism ◽  
Dimensional Structure ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Novel Target ◽  
Rate Limiting

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize γ-32P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

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