scholarly journals Inhibition patterns of a model complex mimicking the reductive half-reaction of sulphite oxidase

1996 ◽  
Vol 319 (3) ◽  
pp. 953-959 ◽  
Author(s):  
Pradeep K CHAUDHURY ◽  
Samar K DAS ◽  
Sabyasachi SARKAR
Keyword(s):  
Steady State ◽  
Ternary Complex ◽  
Mixed Type ◽  
Catalytic Site ◽  
Binary Complex ◽  
Allosteric Site ◽  
Equilibrium Conditions ◽  
Enzyme Substrate ◽  
Sulphite Oxidase ◽  
Model Complex

Different inhibition types of the saturation kinetics involving a synthesized model complex, [Bu4N]2[MoVIO2(mnt)2] (E) (where mnt2- = 1,2-dicyanoethylenedithiolate), and HSO3- as the substrate (S) by structurally similar anions SO42-, H2PO4- and H2PO3- have been shown for the first time in relevance to the reductive half reaction of the native enzyme sulphite oxidase. SO42- acts as a competitive inhibitor. The mixed-type non-competitive inhibition by H2PO4- and the sigmoidal-type inhibition by H2 PO3- are explained by a diamond-configuration random-order model. This involves a random binding sequence of the substrate and the inhibitor, and forms, in addition to two binary complexes [enzyme-substrate (ES) and enzyme-inhibitor (EI)], one enzyme-substrate-inhibitor-type ternary complex (ESI) by participation of at least one more binding site in addition to the catalytic site. This is possible in the present case only by co-ordination enhancement of molybdenum in E. This co-ordination expansion is brought about by nucleophilic attack of the substrate or the inhibitor at the molybdenum, forming a hepta-coordinated binary complex with the generation of an oxoanionic functional site, called the allosteric site. Analysis of the experimental data suggests that the inhibition by H2PO4- is due to the mechanism following either equilibrium conditions or a combination of steady-state and equilibrium conditions. With H2PO3-, the inhibition is due to the mechanism following the steady-state conditions. It is also shown that the ternary complex involving the enzyme, substrate and H2PO4- or H2 PO3- is productive, but at a lower rate than that of the enzyme-substrate binary complex. Mixed-type inhibition with H2PO4- is actually of the type called ‘partially mixed competitive and non-competitive’ as the inhibitor binds both to the catalytic site and to the allosteric site. The sigmoidal-type inhibition by H2PO3- is similar to heterotropic allosteric effect of mixed V,K type with the distinction, however, that the significance of co-operativity in this case is of kinetic importance only. Received 3 January 1996/20 May 1996; accepted 25 June 1996

scholarly journals Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 477-484
Author(s):  
W F Wu ◽  
S Christiansen ◽  
M Feiss
Keyword(s):  
Protein Interactions ◽  
Ternary Complex ◽  
Large Subunit ◽  
Small Subunit ◽  
Functional Domain ◽  
Binary Complex ◽  
Protein Protein Interactions ◽  
Related Phage ◽  
Carboxy Terminal ◽  
Lambda Dna

Abstract The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase. Terminase binds to lambda DNA at cosB to form a binary complex. The terminase:DNA complex binds a prohead to form a ternary complex. Ternary complex formation involves an interaction of the prohead with gpA. The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding. This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21. lambda and 21 encode terminases that are analogous in structural organization and have ca. 60% sequence identity. In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding. A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities. In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized. lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene. The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene. The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Takeo Tomita ◽  
Hajime Matsushita ◽  
Ayako Yoshida ◽  
Saori Kosono ◽  
Minoru Yoshida ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Glutamate Dehydrogenase ◽  
Ternary Complex ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Regulatory Subunit ◽  
Binary Complex ◽  
Bacterial Cells ◽  
Adenine Phosphoribosyltransferase ◽  
Content Type

ABSTRACT Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells. IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

Fluorescence Enhancement of Europium(III) Perchlorate by 2,2′-Dipyridyl on Bis(benzylsulfinyl)methane Complex

2013 ◽  
Vol 634-638 ◽  
pp. 2462-2465
Author(s):  
Wen Xian Li ◽  
Bo Yang Ao ◽  
Jing Zhang
Keyword(s):  
Rare Earth ◽  
Ternary Complex ◽  
Fluorescence Enhancement ◽  
Fluorescence Emission ◽  
Europium Complex ◽  
New Method ◽  
Rare Earth Complexes ◽  
Fluorescence Properties ◽  
Binary Complex ◽  
Fluorescence Lifetimes

A novel ligand with double sulfinyl groups, bis(benzylsulfinyl)methane L, was synthesized by a new method. Its novel ternary complex, has been synthesized [using L as the first ligand, and dipyridyl L' as the second ligand]. In order to study the effect of the second ligand on the fluorescence properties of rare-earth sulfoxide complex, a novel binary europium complex has been synthesized. Photoluminescent measurement showed that the first ligand L could efficiently transfer the energy to Eu (III) ions in the complex. Furthermore, the detailed luminescence analyses on the rare earth complexes indicated that the ternary Eu (III) complex manifested stronger fluorescence intensities, longer lifetimes, and higher fluorescence quantum efficiencies than the binary Eu (III) materials. The fluorescence emission intensities and fluorescence lifetimes of the ternary complex enhanced more obviously than the binary complex.

THEORY OF THE TRANSIENT PHASE IN AN ENZYME SYSTEM INVOLVING TWO ENZYME-SUBSTRATE COMPLEXES: THE CASE OF THE FORMATION OF PRODUCTS FROM THE FIRST COMPLEX

1959 ◽  
Vol 37 (4) ◽  
pp. 737-743 ◽  
Author(s):  
Ludovic Ouellet ◽  
James A. Stewart
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Active Site ◽  
Substrate Concentration ◽  
Enzyme System ◽  
Kinetic Scheme ◽  
Enzyme Concentration ◽  
Theoretical Treatment ◽  
Transient Phase ◽  
Enzyme Substrate

A theoretical treatment is worked out for the kinetic scheme[Formula: see text]in which the concentration of P1 is followed. The steady-state and transient phase equations are obtained subject to the condition that the substrate concentration is greatly in excess of the enzyme concentration. The conditions under which evidence in favor of this mechanism can be obtained from experimental data are discussed. Under certain conditions, the weight of the enzyme corresponding to one active site can be determined. Methods for the evaluation of the different constants are described.

Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

2013 ◽  
Vol 450 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Debajyoti Dutta ◽  
Sudipta Bhattacharyya ◽  
Amlan Roychowdhury ◽  
Rupam Biswas ◽  
Amit Kumar Das
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Ternary Complex ◽  
Catalytic Mechanism ◽  
Acyl Carrier Protein ◽  
Structural Data ◽  
Acid Synthesis ◽  
Binary Complex ◽  
C Terminus ◽  
Covalently Linked

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

scholarly journals Sorption/Diffusion Contributions to the Gas Permeation Properties of Bi-Soft Segment Polyurethane/Polycaprolactone Membranes for Membrane Blood Oxygenators

Membranes ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 8
Author(s):  
Tiago M. Eusébio ◽  
Ana Rita Martins ◽  
Gabriela Pon ◽  
Mónica Faria ◽  
Pedro Morgado ◽  
...  
Keyword(s):  
Steady State ◽  
Soft Segment ◽  
Time Lag ◽  
Transient State ◽  
Gas Permeation ◽  
Dominant Factor ◽  
Atmospheric Gases ◽  
Equilibrium Conditions ◽  
Permeability Coefficients ◽  
Permeation Properties

Due to their high hemocompatibility and gas permeation capacity, bi-soft segment polyurethane/polycaprolactone (PU/PCL) polymers are promising materials for use in membrane blood oxygenators. In this work, both nonporous symmetric and integral asymmetric PU/PCL membranes were synthesized, and the permeation properties of the atmospheric gases N2, O2, and CO2 through these membranes were experimentally determined using a new custom-built gas permeation apparatus. Permeate pressure vs. time curves were obtained at 37.0 °C and gas feed pressures up to 5 bar. Fluxes, permeances, and permeability coefficients were determined from the steady-state part of the curves, and the diffusion and sorption coefficients were estimated from the analysis of the transient state using the time-lag method. Independent measurements of the sorption coefficients of the three gases were performed, under equilibrium conditions, in order to validate the new setup and procedure. This work shows that the gas sorption in the PU/PCL polymers is the dominant factor for the permeation properties of the atmospheric gases in these membranes.

Transient-Phase and Steady-State Kinetics for Enzyme Activation

1973 ◽  
Vol 51 (6) ◽  
pp. 806-814 ◽  
Author(s):  
Nasrat H. Hijazi ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Kinetic Data ◽  
Enzyme Activation ◽  
Transient Phase ◽  
Time Curve ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Substrate Activation ◽  
Steady State Kinetics ◽  
Special Case

A non-steady-state analysis has been worked out for two mechanisms in which an activator Q can become attached to an enzyme–substrate complex EA, the species EAQ breaking down more rapidly than EA. It is shown that if EAQ breaks down into EQ + product there can be no steady state. If, however, EAQ breaks down into E + Q + product, the transient phase is followed by a steady state in which the product versus time curve is linear. A special case of this mechanism is when Q is the substrate (substrate activation). Some published kinetic data on carboxypeptidase are analyzed with reference to the equations derived.

Refined structure of the FKBP12–rapamycin–FRB ternary complex at 2.2 Å resolution

1999 ◽  
Vol 55 (4) ◽  
pp. 736-744 ◽  
Author(s):  
Jun Liang ◽  
Jungwon Choi ◽  
Jon Clardy
Keyword(s):  
Cell Cycle ◽  
Biological Activity ◽  
High Resolution ◽  
Protein Interactions ◽  
Ternary Complex ◽  
Binary Complex ◽  
Fk506 Binding Protein ◽  
Cycle Arrest ◽  
Protein Partners

The structure of the FKBP12–rapamycin–FRB ternary complex has now been refined at 2.2 Å resolution. The cell-cycle arrest agent rapamycin binds FK506-binding protein (FKBP12) and the FKBP12–rapamycin binding (FRB) domain of FKBP12–rapamycin associated protein (FRAP) simultaneously, and the inhibition of FRAP is responsible for rapamycin's biological activity. The conformation of rapamycin in the ternary complex is very similar to that observed in the FKBP12–rapamycin binary complex, with an r.m.s. difference of only 0.30 Å. However, a slight (9°) rotation repositions the FRB-binding face of rapamycin in the ternary complex. There are extensive rapamycin–protein interactions and relatively few interactions between the two protein partners FKBP12 and FRB, these interactions mainly involving residues in the 40s and 80s loops of FKBP12 and α1 and α4 of FRB. The high-resolution refinement has revealed the crucial role of several buried waters in the formation of the ternary complex.

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