scholarly journals The interaction of troponin C with phosphofructokinase. Comparison with calmodulin

1991 ◽  
Vol 274 (2) ◽  
pp. 445-451 ◽  
Author(s):  
J Lan ◽  
R F Steiner
Keyword(s):  
Catalytic Activity ◽  
Light Scattering ◽  
Dissociation Constant ◽  
Conformational Change ◽  
Conformational Changes ◽  
Troponin C ◽  
Binary Complex ◽  
Fluorescent Labels ◽  
Apparent Dissociation Constant ◽  
Intact Molecule

Phosphofructokinase (PFK) is a calmodulin (CaM)-binding protein [Mayr & Heilmeyer (1983) FEBS Lett. 195, 51-57]. We found that troponin C (TnC), which is homologous to CaM, also binds PFK and affects PFK's catalytic activity, aggregation states and conformational changes as CaM does in most cases. PFK titration of N-acetylaminoethyl-5-naphthylamido-1-sulphonate (‘AEDANS’)-TnC showed that its apparent dissociation constant is comparable with that of PFK-CaM. Fluorescent labels were also used to probe contact regions on TnC and CaM. It is likely that the C-terminal end of the connecting strand of the TnC molecule is close to PFK in the binary complex. Hydrophobic regions of TnC and CaM also possibly play roles in the binding and polymerization of PFK. TnC and CaM deactivate PFK through accelerating PFK conformational change as well as through accelerating PFK tetramer dissociation, as implied in the results of activity, light-scattering, fluorescence and c.d. experiments. The intact molecule of CaM appears to be required to deactivate PFK, because neither half of the CaM molecule has an effect on PFK activity.

Substrate-binding recognition and specificity of trehalose phosphorylase from Schizophyllum commune examined in steady-state kinetic studies with deoxy and deoxyfluoro substrate analogues and inhibitors

2002 ◽  
Vol 363 (2) ◽  
pp. 335-340 ◽  
Author(s):  
Christian EIS ◽  
Bernd NIDETZKY
Keyword(s):  
Hydrogen Bonding ◽  
Dissociation Constant ◽  
Ternary Complex ◽  
Tight Binding ◽  
Schizophyllum Commune ◽  
Hydroxyl Groups ◽  
Binary Complex ◽  
Apparent Dissociation Constant ◽  
Leaving Group ◽  
Trehalose Phosphorylase

Trehalose phosphorylase is a component of the α-d-glucopyranosyl α-d-glucopyranoside (α,α-trehalose)-degrading enzyme system in fungi and it catalyses glucosyl transfer from α,α-trehalose to phosphate with net retention of the anomeric configuration. The enzyme active site has no detectable affinity for α,α-trehalose in the absence of bound phosphate and catalysis occurs from the ternary complex. To examine the role of non-covalent enzyme—substrate interactions for trehalose phosphorylase recognition, we used the purified enzyme from Schizophyllum commune and tested a series of incompetent structural analogues of the natural substrates and products as inhibitors of the enzyme. Equilibrium-binding constants (Ki) for deoxy- and deoxyfluoro derivatives of d-glucose show that loss of interactions with the 3-, 4- or 6-OH, but not the reactive 1- and the 2-OH, results in considerably (≥100-fold) weaker affinity for sugar-binding subsite +1, revealing the requirement for hydrogen bonding with hydroxyls, away from the site of chemical transformation to position precisely the d-glucose-leaving group/nucleophile for catalysis. The high specificity of trehalose phosphorylase for the sugar aglycon during binding and conversion of O-glycosides is in contrast with the observed α-retaining phosphorolysis of α-d-glucose-1-fluoride (α-d-Glc-1-F) since the productive bonding capability of the fluoride-leaving group with subsite +1 is minimal. The specificity constant (19M−1·s−1) and catalytic-centre activity (0.1s−1) for the reaction with α-d-Glc-1-F are 0.10- and 0.008-fold the corresponding kinetic parameters for the enzymic reaction with α,α-trehalose. The non-selective-inhibition profile for a series of inactive α-d-glycopyranosyl phosphates shows that the driving force for the binary-complex formation lies mainly in interactions of the enzyme with the phosphate group and suggests that hydrogen bonding with hydroxyl groups at the catalytic site (subsite −1) contributes to catalysis by providing stabilization, which is specific to the transition state. Vanadate, a tight-binding phosphate mimic, inhibits the phosphorolysis of α-d-Glc-1-F by forming a ternary complex whose apparent dissociation constant of 120μM is approx. 160-fold greater than the dissociation constant of the same inhibitor complex with α,α-trehalose.

scholarly journals Linking Bi-Metal Distribution Patterns in Porous Carbon Nitride Fullerene to Its Catalytic Activity toward Gas Adsorption

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1794
Author(s):  
Parisa Nematollahi ◽  
Erik C. Neyts
Keyword(s):  
Catalytic Activity ◽  
Carbon Nitride ◽  
Gas Adsorption ◽  
Distribution Patterns ◽  
Catalyst Design ◽  
Metal Distribution ◽  
Binary Complex ◽  
Metal Atoms ◽  
Metal Composition ◽  
Single Transition

Immobilization of two single transition metal (TM) atoms on a substrate host opens numerous possibilities for catalyst design. If the substrate contains more than one vacancy site, the combination of TMs along with their distribution patterns becomes a design parameter potentially complementary to the substrate itself and the bi-metal composition. By means of DFT calculations, we modeled three dissimilar bi-metal atoms (Ti, Mn, and Cu) doped into the six porphyrin-like cavities of porous C24N24 fullerene, considering different bi-metal distribution patterns for each binary complex, viz. TixCuz@C24N24, TixMny@C24N24, and MnyCuz@C24N24 (with x, y, z = 0–6). We elucidate whether controlling the distribution of bi-metal atoms into the C24N24 cavities can alter their catalytic activity toward CO2, NO2, H2, and N2 gas capture. Interestingly, Ti2Mn4@C24N24 and Ti2Cu4@C24N24 complexes showed the highest activity and selectively toward gas capture. Our findings provide useful information for further design of novel few-atom carbon-nitride-based catalysts.

scholarly journals New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White
Keyword(s):  
Influenza Virus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

Binding of adenylosuccinate synthetase to contractile proteins of muscle

1989 ◽  
Vol 257 (1) ◽  
pp. C29-C35 ◽  
Author(s):  
J. P. Manfredi ◽  
R. Marquetant ◽  
A. D. Magid ◽  
E. W. Holmes
Keyword(s):  
Ionic Strength ◽  
Dissociation Constant ◽  
Thin Filament ◽  
Contractile Proteins ◽  
Purine Nucleotide ◽  
Apparent Dissociation Constant ◽  
Thin Filaments ◽  
Adenylosuccinate Synthetase ◽  
Purine Nucleotide Cycle ◽  
Low Ionic Strength

The muscle isozyme of adenylosuccinate synthetase (AdSS), an enzyme of the purine nucleotide cycle, has previously been shown to bind to purified F-actin in buffers of low ionic strength and pH (Ogawa et al. Eur. J. Biochem. 85: 331-338, 1978). We have extended these observations by measuring the association of both crude and purified AdSS with the contractile proteins of muscle in buffers of physiological ionic strength and pH. Under these conditions, the enzyme binds to F-actin, actin-tropomyosin complexes, reconstructed thin filaments, and myofibrils but not to myosin. The apparent dissociation constant of 1.2 microM and binding maximum of 2.6 nmol enzyme/mg myofibrils indicate that binding of AdSS to myofibrils can be physiologically significant. The results suggest that AdSS in muscle may be associated with the thin filament of myofibrils.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

2018 ◽  
Vol 118 (02) ◽  
pp. 340-350 ◽  
Author(s):  
Ingrid Stroo ◽  
J. Marquart ◽  
Kamran Bakhtiari ◽  
Tom Plug ◽  
Alexander Meijer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Active Form ◽  
Coagulation Factor ◽  
Lysine Residue ◽  
Factor Ix ◽  
Factor Xi ◽  
Lysine Residues ◽  
Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

scholarly journals Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

1974 ◽  
Vol 141 (2) ◽  
pp. 365-381 ◽  
Author(s):  
Christopher W. Wharton ◽  
Athel Cornish-Bowden ◽  
Keith Brocklehurst ◽  
Eric M. Crook
Keyword(s):  
Methyl Ester ◽  
Dissociation Constant ◽  
Computational Methods ◽  
Rate Constant ◽  
Guanidine Hydrochloride ◽  
Ester Hydrolysis ◽  
Binary Complex ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Ester Substrate

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10-2±0.32×10-2s-1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s-1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s-1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.

The Physiology of Carbon Dioxide Transport in Insect Blood

1950 ◽  
Vol 27 (2) ◽  
pp. 158-174 ◽  
Author(s):  
L. LEVENBOOK
Keyword(s):  
Carbon Dioxide ◽  
Dissociation Constant ◽  
Temperature Coefficient ◽  
Carbonic Acid ◽  
Apparent Dissociation Constant ◽  
Carbon Dioxide Transport ◽  
High Co2 ◽  
The Third ◽  
A Value ◽  
Dissociation Curves

1. The pH of the blood of the third instar Gastrophilus larva is 6.64 at 38° C. with a pH-temperature coefficient of -0.007 Per 1° C. rise in temperature. 2. The total CO2 content of the blood varies from 40.6 to 131.4 vol. % with an average of 72.4 vol. %. The CO2 content of the tissues minus the cuticle is very close to, and follows the variations in, the CO2 content of the blood. 3. The CO2 tension in the blood is from 300 to 500 mm. Hg. From 30 to 50% of the CO2 is in solution, the rest in the form of bicarbonate. Carbamate formation does not occur in the blood. 4. The ‘apparent’ dissociation constant for carbonic acid, (pK'1), has a value of 6.08 (S.D. ±0.06) at 38° C. and 6.19 (s.d. ±0.13) at 16° C. 5. CO2 dissociation curves have been drawn for 38 and 16° C. The slope of the curves indicates that the whole of the CO2 is given off at zero CO2 tension, and that the blood is adapted for functioning at high CO2 tensions.

Characterization of a Hyperpolarization-Activated Inward Current in Cajal-Retzius Cells in Rat Neonatal Neocortex

2000 ◽  
Vol 84 (3) ◽  
pp. 1681-1691 ◽  
Author(s):  
Werner Kilb ◽  
Heiko J. Luhmann
Keyword(s):  
Dissociation Constant ◽  
Reversal Potential ◽  
Hill Coefficient ◽  
Resting Membrane Potential ◽  
Patch Clamp Technique ◽  
Apparent Dissociation Constant ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Inward Current ◽  
Retzius Cells

Cajal-Retzius cells are among the first neurons appearing during corticogenesis and play an important role in the establishment of cortical lamination. To characterize the hyperpolarization-activated inward current ( I h) and to investigate whether I h contributes to the relatively positive resting membrane potential (RMP) of these cells, we analyzed the properties of I h in visually identified Cajal-Retzius cells in cortical slices from neonatal rats using the whole cell patch-clamp technique. Membrane hyperpolarization to −90 mV activated a prominent inward current that was inhibited by 1 mM Cs+ and was insensitive to 1 mM Ba2+. The activation time constant for I h was strongly voltage dependent. In Na+-free solution, I h was reduced, indicating a contribution of Na+. An analysis of the tail currents revealed a reversal potential of −45.2 mV, corresponding to a permeability coefficient (pNa+/pK+) of 0.13. While an increase in the extracellular K+ concentration ([K+]e) enhances I h, it was reduced by a [K+]e decrease. This [K+]e dependence could not be explained by an effect on the electromotive force on K+ but suggested an additional extracellular binding site for K+ with an apparent dissociation constant of 7.2 mM. Complete Cl−substitution by Br−, I−, or NO3 − had no significant effect on I h, whereas a complete Cl−substitution by the organic compounds methylsulfate, isethionate, or gluconate reduced I h by ∼40%. The I h reduction observed in gluconate could be abolished by the addition of Cl−. The analysis of the [Cl−]e dependence of I h revealed a dissociation constant of 9.8 mM and a Hill-coefficient of 2.5, while the assumption of a gluconate-dependent I h reduction required an unreasonably high Hill-coefficient >20. An internal perfusion with the lidocaine derivative lidocaine N-ethyl bromide blocks I h within 1 min after establishment of the whole cell configuration. An inhibition of I h by 1 mM Cs+ was without an effect on RMP, action potential amplitude, threshold, width, or afterhyperpolarization. We conclude from these results that Cajal-Retzius cells express a prominent I hwith characteristic properties that does not contribute to the RMP.

Interaction Of Fibrin And Tissue Plasminogen Activator

1981 ◽  
Author(s):  
P Wallén ◽  
M Rånby
Keyword(s):  
Dissociation Constant ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Conformational Changes ◽  
Specific Interaction ◽  
Kinetic Studies ◽  
Test System ◽  
Activation Rate ◽  
Tissue Plasminogen ◽  
Analytical System

Fibrin itself has a marked influence on fibrinolysis induced by tissue plasminogen activator (TA) indicating a specific interaction. The interaction between fibrin and TA is manifested in two ways, 1) a marked stimulating effect of fibrin on the activation of plasminogen; 2) physical ad- sorbtion of TA on fibrin. By measurement in a sensitive analytical system in which the generation of plasmin is followed by a chromogenic substrate it has been shown that TA is a rather poor activator of plasminogen. In the presence of fibrin the kinetics of the activation is dramatically changed. A stimulation up to 1000-fold is obtained at low plasminogen concentrations. As for activation of native plasminogen (Glu-plasminogen) there is a decrease of km (about 15-fold) as well as an increase of kc (about 80-fold). Fibrinogen has comparatively little effect on the activation rate (at the most 10-fold). The amidolytic activity of TA, using an activator sensitive substrate, is not influenced by fibrin indicating that the effect is not due to conformational changes in the active site region of TA.By varying the concentration of fibrin in the test system it has been demonstrated that the stimulation effect increases suddenly at a fibrin concentration of about 0.01μM. It was suggested that this value represents the dissociation constant of the TA-fibrin complex. However, the amount of fibrin necessary for the adsorbtion of TA in purification experiments indicates a significantly higher dissociation constant (about 0.4 μM). An important difference between the purification experiments and the studies on fibrin stimulation is that plasminogen (plasmin) is absent in the former studies. The formation of a triple complex between fibrin, plasminogen and TA may be the explanation for a more efficient binding of TA in the kinetic studies.

Sign in / Sign up

Export Citation Format

Share Document