scholarly journals Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin

1988 ◽  
Vol 251 (2) ◽  
pp. 515-526 ◽  
Author(s):  
A P Jackson ◽  
C R Bagshaw
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Kinetic Studies ◽  
Tryptophan Fluorescence ◽  
Adductor Muscle ◽  
Reverse Direction ◽  
Heavy Meromyosin ◽  
Thin Filaments ◽  
Adp Release ◽  
Atp Binding And Hydrolysis

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.

scholarly journals Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

2021 ◽  
Author(s):  
Alessandro Borsellini ◽  
Vladislav Kunetsky ◽  
Peter Friedhoff ◽  
Meindert H. Lamers
Keyword(s):  
Dna Binding ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dna Mismatch Repair ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Adp Release ◽  
Atp Binding And Hydrolysis

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

scholarly journals Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2

2020 ◽  
Author(s):  
Alexander V Peskin ◽  
Flávia C Meotti ◽  
Luiz F de Souza ◽  
Robert F Anderson ◽  
Christine C Winterbourn ◽  
...  
Keyword(s):  
Active Site ◽  
Disulfide Bonds ◽  
Flow Analysis ◽  
Kinetic Studies ◽  
Tryptophan Fluorescence ◽  
Negative Cooperativity ◽  
Sulfenic Acid ◽  
Peroxiredoxin 2 ◽  
Active Site Cysteine ◽  
Sds Page

ABSTRACTPeroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 ± 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 ± 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 ± 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.

scholarly journals Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes

1985 ◽  
Vol 225 (1) ◽  
pp. 209-217 ◽  
Author(s):  
E T Bell ◽  
C LiMuti ◽  
C L Renz ◽  
J E Bell
Keyword(s):  
Glutamate Dehydrogenase ◽  
Conformational Changes ◽  
Active Site ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Fluorescence Properties ◽  
Subunit Interactions ◽  
Oxidative Deamination ◽  
Substrate Analogues

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.

scholarly journals Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides

1988 ◽  
Vol 251 (2) ◽  
pp. 527-540 ◽  
Author(s):  
A P Jackson ◽  
C R Bagshaw
Keyword(s):  
Conformational Changes ◽  
Rate Constants ◽  
Tryptophan Fluorescence ◽  
Fluorescence Decay ◽  
Fluorescence Signal ◽  
Heavy Meromyosin ◽  
Protein Fluorescence ◽  
Association Rate Constants ◽  
Kinetic Trapping ◽  
Kinetics Of

The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.

pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

2019 ◽  
Vol 26 (10) ◽  
pp. 743-750 ◽  
Author(s):  
Remya Radha ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Kinetic Studies ◽  
Structural Features ◽  
Structure Stability ◽  
Kcal Mole ◽  
Scanning Calorimetry ◽  
Wide Range ◽  
Ph Dependent ◽  
Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

scholarly journals Active site lysine backbone undergoes conformational changes in the bacteriorhodopsin photocycle.

1994 ◽  
Vol 269 (10) ◽  
pp. 7387-7389
Author(s):  
H. Takei ◽  
Y. Gat ◽  
Z. Rothman ◽  
A. Lewis ◽  
M. Sheves
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Bacteriorhodopsin Photocycle

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

scholarly journals A structural framework for unidirectional transport by a bacterial ABC exporter

2020 ◽  
Vol 117 (32) ◽  
pp. 19228-19236
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Conformational Changes ◽  
Iron Homeostasis ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Reverse Direction ◽  
Conformational States ◽  
X Ray Crystallography ◽  
Binding Domains ◽  
Structural Framework ◽  
Glutathione Derivatives

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog fromNovosphingobium aromaticivorans(NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying theNaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. AsNaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport byNaAtm1. One of the disulfide crosslinkedNaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

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