Temperature Sensitivity of Myosin and Actomyosin

1973 ◽  
Vol 51 (1) ◽  
pp. 71-86 ◽  
Author(s):  
A. L. Jacobson ◽  
J. Henderson
Keyword(s):  
Enzymatic Activity ◽  
Conformational Changes ◽  
Temperature Sensitivity ◽  
Active Site ◽  
Temperature Effects ◽  
Thermal Denaturation ◽  
Site Analysis ◽  
Maximum Change ◽  
Temperature Inactivation

The thermal denaturation of myosin and actomyosin was studied by active site analysis (enzymatic activity) and measurements were related to overall conformational changes (viscosity) over the temperature range 19–65 °C. The role of sulfhydryl (SH) groups on the temperature-induced denaturation of actomyosin was investigated. The temperature of maximum change in the overall conformation (the melting temperature, TM) was unaffected by the binding of F-actin to myosin. For both myosin and actomyosin the TM was 43 ± 2 °C. However, the range of temperature over which large conformational changes were observed was affected by the binding of F-actin to myosin. For myosin and dissociated actomyosin, changes were observed between 37 and 50 °C, while for actomyosin large changes were observed between 20 and 50 °C. With actomyosin there was an irreversible increase in titratable sulfhydryl groups from 19 to 60 °C. Temperature effects on the calcium-activated ATPase were studied. The temperature of maximum enzymatic activity for actomyosin was 45 ± 2 °C, which corresponds to the TM and the temperature at which increases in SH content were apparent. However, for myosin the temperature of maximum enzymatic activity was 33 °C, considerably below the TM. Overall conformational changes were reversible below the TM, while changes in enzymatic activity were reversible below the temperature of maximum enzymatic activity. Actin offers considerable protection to the temperature inactivation of the active site of myosin even though the F-actin–myosin complex is very highly dissociated in the presence of ATP. However, there is no significant stabilization of myosin by F-actin in terms of the temperature sensitivity of the overall conformation.

scholarly journals The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism

1998 ◽  
Vol 333 (3) ◽  
pp. 811-816 ◽  
Author(s):  
Antonio PÁRRAGA ◽  
Isabel GARCÍA-SÁEZ ◽  
Sinead B. WALSH ◽  
Timothy J. MANTLE ◽  
Miquel COLL
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Water Molecules ◽  
X Ray Diffraction ◽  
Glutathione S Transferase ◽  
X Ray ◽  
The Right

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme–GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

scholarly journals Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Nelson P. Guerra ◽  
Lorenzo Pastrana Castro
Keyword(s):  
Enzymatic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Ageing Time ◽  
Enzymatic Reactions ◽  
Reaction Products ◽  
Inhibitory Effects ◽  
Amylolytic Enzymes ◽  
Modified Forms

The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics,vmaxdecreased significantly (P<0.05) andKMincreased (although not always significantly) with the increase int. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects oftand [G].

Structural insights into the peroxidase activity and inactivation of human peroxiredoxin 4

2011 ◽  
Vol 441 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Xi Wang ◽  
Likun Wang ◽  
Xi'e Wang ◽  
Fei Sun ◽  
Chih-chen Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Analysis ◽  
Conformational Changes ◽  
Active Site ◽  
High Reactivity ◽  
Structural Explanation ◽  
Disulfide Formation ◽  
Redox Forms ◽  
Structural Insights

Prx4 (peroxiredoxin 4) is the only peroxiredoxin located in the ER (endoplasmic reticulum) and a proposed scavenger for H2O2. In the present study, we solved crystal structures of human Prx4 in three different redox forms and characterized the reaction features of Prx4 with H2O2. Prx4 exhibits a toroid-shaped decamer constructed of five catalytic dimers. Structural analysis revealed conformational changes around helix α2 and the C-terminal reigon with a YF (Tyr-Phe) motif from the partner subunit, which are required for interchain disulfide formation between Cys87 and Cys208, a critical step of the catalysis. The structural explanation for the restricting role of the YF motif on the active site dynamics is provided in detail. Prx4 has a high reactivity with H2O2, but is susceptible to overoxidation and consequent inactivation by H2O2. Either deletion of the YF motif or dissociation into dimers decreased the susceptibility of Prx4 to overoxidation by increasing the flexibility of Cys87.

scholarly journals Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

2000 ◽  
Vol 346 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Richard GRIEßLER ◽  
Sabato D'AURIA ◽  
Reinhard SCHINZEL ◽  
Fabio TANFANI ◽  
Bernd NIDETZKY
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Active Site ◽  
Thermal Denaturation ◽  
Hydrophobic Interactions ◽  
Wild Type ◽  
Reversible Inactivation ◽  
The Stability ◽  
Maltodextrin Phosphorylase

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each ≈ 90-kDa subunit contains active-site pyridoxal 5ʹ-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133 → Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at ≈ 45 °C. They are manifested by slight increases in unordered structure and 1H/2H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at ≈ 45 °C without prior release into solution of pyridoxal 5ʹ-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 °C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 °C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a ≈ 15 °C higher value on the temperature scale. Apart from greater stability, the Asn-133 → Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.

scholarly journals Molecular Insights on exquisitely Selective SrtA inhibitors towards active site loop forming open/close lid conformations in SrtA from Bacillus anthracis

2019 ◽  
Author(s):  
Chandrabose Selvaraj ◽  
Gurudeeban Selvaraj ◽  
Satyavani Kaliamurthi ◽  
Dong-Qing Wei ◽  
Sanjeev Kumar Singh
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Conformational Changes ◽  
Active Site ◽  
3D Qsar ◽  
Sortase A ◽  
Protein Conformational Changes ◽  
A Cell ◽  
Inhibitory Activities

AbstractThe present study clearly explains the dependency of inhibitory activities in SrtA inhibitors is closely related to protein conformational changes of SrtA from Bacillus anthracis B. anthracisSortase A (SrtA) protein anchors proteins by recognizing a cell wall sorting signal containing the amino acid sequence LPXTG In order to analyze conformational changes and the role of SrtA enzyme, especially the loop motions which situated proximal to the active site molecular dynamic simulation was carried out for 100ns. Particular loop is examined for its various conformations from the MD trajectories and the open/close lid conformations are considered for the enzyme activity validations. Experimentally verified SrtA inhibitors activity was analyzed through 3D-QSAR and Molecular docking approaches. Results indicate that, biological activity of SrtA inhibitors is closely related to the closed lid conformation of SrtA from Bacillus anthracis. This work may lead to a better understanding of the mechanism of action and aid to design a novel and more potent SrtA inhibitors.

scholarly journals Comment on: Role of changes in the L3 loop of the active site in the evolution of enzymatic activity of VIM-type metallo- -lactamases

2010 ◽  
Vol 66 (3) ◽  
pp. 684-685 ◽  
Author(s):  
M. Castanheira ◽  
L. M. Deshpande ◽  
R. E. Mendes ◽  
E. Rodriguez-Noriega ◽  
R. N. Jones ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Active Site

Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues

1977 ◽  
Vol 55 (8) ◽  
pp. 856-864 ◽  
Author(s):  
T. J. Carne ◽  
T. G. Flynn
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Enzyme Inactivation ◽  
Breast Muscle ◽  
Chicken Breast ◽  
Chemically Modified ◽  
Activity Loss ◽  
Lysyl Residue

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5′-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff s base complex between pyridoxal 5′-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5′-phosphate per mole of PGM dimer through the ε-amino group of a lysyl residue. The effect of pyridoxal 5′-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5′-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5′-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5′-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme.The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.

scholarly journals Active-Site Conformational Fluctuations Promote the Enzymatic Activity of NDM-1

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Hongmin Zhang ◽  
Guixing Ma ◽  
Yifan Zhu ◽  
Lingxiao Zeng ◽  
Ashfaq Ahmad ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Bacterial Infections ◽  
Site Directed Mutagenesis ◽  
New Delhi ◽  
Native Form ◽  
Conformational Fluctuation ◽  
Almost All

ABSTRACT β-Lactam antibiotics are the mainstay for the treatment of bacterial infections. However, elevated resistance to these antibiotics mediated by metallo-β-lactamases (MBLs) has become a global concern. New Delhi metallo-β-lactamase-1 (NDM-1), a newly added member of the MBL family that can hydrolyze almost all β-lactam antibiotics, has rapidly spread all over the world and poses serious clinical threats. Broad-spectrum and mechanism-based inhibitors against all MBLs are highly desired, but the differential mechanisms of MBLs toward different antibiotics pose a great challenge. To facilitate the design of mechanism-based inhibitors, we investigated the active-site conformational changes of NDM-1 through the determination of a series of 15 high-resolution crystal structures in native form and in complex with products and by using biochemical and biophysical studies, site-directed mutagenesis, and molecular dynamics computation. The structural studies reveal the consistency of the active-site conformations in NDM-1/product complexes and the fluctuation in native NDM-1 structures. The enzymatic measurements indicate a correlation between enzymatic activity and the active-site fluctuation, with more fluctuation favoring higher activity. This correlation is further validated by structural and enzymatic studies of the Q123G mutant. Our combinational studies suggest that active-site conformational fluctuation promotes the enzymatic activity of NDM-1, which may guide further mechanism studies and inhibitor design.

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