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 Cwp84, aClostridium difficilecysteine protease, exhibits conformational flexibility in the absence of its propeptide

2015 ◽  
Vol 71 (3) ◽  
pp. 295-303 ◽  
Author(s):  
William J. Bradshaw ◽  
April K. Roberts ◽  
Clifford C. Shone ◽  
K. Ravi Acharya
Keyword(s):  
Molecular Weight ◽  
Conformational Changes ◽  
Active Site ◽  
Conformational Flexibility ◽  
Low Molecular Weight ◽  
Antibiotic Resistant ◽  
Multiple Copies ◽  
Antibiotic Resistant Bacterium ◽  
Structural Insights

In recent decades, the global healthcare problems caused byClostridium difficilehave increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fightingC. difficileinfections. The surface layer (S-layer) ofC. difficileis likely to be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket.

scholarly journals Characterization of the endoplasmic reticulum–resident peroxidases GPx7 and GPx8 shows the higher oxidative activity of GPx7 and its linkage to oxidative protein folding

2020 ◽  
Vol 295 (36) ◽  
pp. 12772-12785 ◽  
Author(s):  
Shingo Kanemura ◽  
Elza Firdiani Sofia ◽  
Naoya Hirai ◽  
Masaki Okumura ◽  
Hiroshi Kadokura ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Physiological Role ◽  
High Reactivity ◽  
Oxidation Activity ◽  
Formation Pathway ◽  
Oxidative Protein Folding ◽  
Redox Active ◽  
Protein Disulfide

Oxidative protein folding occurs primarily in the mammalian endoplasmic reticulum, enabled by a diverse network comprising more than 20 members of the protein disulfide isomerase (PDI) family and more than five PDI oxidases. Although the canonical disulfide bond formation pathway involving Ero1α and PDI has been well-studied so far, the physiological roles of the newly identified PDI oxidases, glutathione peroxidase-7 (GPx7) and -8 (GPx8), are only poorly understood. We here demonstrated that human GPx7 has much higher reactivity with H2O2 and hence greater PDI oxidation activity than human GPx8. The high reactivity of GPx7 is due to the presence of a catalytic tetrad at the redox-active site, which stabilizes the sulfenylated species generated upon the reaction with H2O2. Although it was previously postulated that GPx7 catalysis involved a highly reactive peroxidatic cysteine that can be sulfenylated by H2O2, we revealed that a resolving cysteine instead regulates the PDI oxidation activity of GPx7. We also determined that GPx7 formed complexes preferentially with PDI and P5 in H2O2-treated cells. Altogether, these results suggest that human GPx7 functions as an H2O2-dependent PDI oxidase in cells, whereas PDI oxidation may not be the central physiological role of human GPx8.

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 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.

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 Probing the role of the conserved residue Glu166 in a class A β-lactamase using neutron and X-ray protein crystallography

2020 ◽  
Vol 76 (2) ◽  
pp. 118-123
Author(s):  
Patricia S. Langan ◽  
Brendan Sullivan ◽  
Kevin L. Weiss ◽  
Leighton Coates
Keyword(s):  
Water Molecule ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Drug Binding ◽  
Bacterial Strains ◽  
Structural Explanation ◽  
X Ray ◽  
Class A ◽  
Kinetic Rates

The amino-acid sequence of the Toho-1 β-lactamase contains several conserved residues in the active site, including Ser70, Lys73, Ser130 and Glu166, some of which coordinate a catalytic water molecule. This catalytic water molecule is essential in the acylation and deacylation parts of the reaction mechanism through which Toho-1 inactivates specific antibiotics and provides resistance to its expressing bacterial strains. To investigate the function of Glu166 in the acylation part of the catalytic mechanism, neutron and X-ray crystallographic studies were performed on a Glu166Gln mutant. The structure of this class A β-lactamase mutant provides several insights into its previously reported reduced drug-binding kinetic rates. A joint refinement of both X-ray and neutron diffraction data was used to study the effects of the Glu166Gln mutation on the active site of Toho-1. This structure reveals that while the Glu166Gln mutation has a somewhat limited impact on the positions of the conserved amino acids within the active site, it displaces the catalytic water molecule from the active site. These subtle changes offer a structural explanation for the previously observed decreases in the binding of non-β-lactam inhibitors such as the recently developed diazobicyclooctane inhibitor avibactam.

Role of oxidative mixed-disulfide formation in elastase–serine proteinase inhibitor (serpin) complex

1996 ◽  
Vol 74 (3) ◽  
pp. 391-401 ◽  
Author(s):  
Suresh C. Tyagi
Keyword(s):  
Conformational Changes ◽  
Proteinase Inhibitor ◽  
Acute Phase Proteins ◽  
Serine Proteinase ◽  
Disulfide Exchange ◽  
Disulfide Formation ◽  
Mixed Disulfide ◽  
Fluorescence Measurements ◽  
Disulfide Linkages

To understand the role of thiol and oxidative mixed-disulfide exchange reaction in serpins, we analyzed the conformation of native and mixed-disulfide forms of α1-proteinase inhibitor (α1PI), α1-antichymotrypsin (α1-ACT), α2-antiplasmin (α2-AP), angiotensinogen, and ovalbumin. The conformation of native and oxidized mixed-disulfide serpins was measured by transverse urea gradient (TUG) gels. The results suggest that the acute phase proteins α1-PI and α1-ACT undergo conformational changes following oxidative mixed-disulfide formation and that α2-AP and angiotensinogen do not. The kinetics of disulfide formation was followed by measuring changes in absorbance at 412 nm resulting from Ellman's reaction of disulfide exchange. The rate of mixed-disulfide formation in albumin was 10-fold faster than in the serpin tested. The rate of disulfide exchange in α1-PI was 2-fold faster than that of α1-ACT. However, disulfide formation in α1-PI and α1-ACT was much slower than for any other serpin, e.g., α2-AP and angiotensinogen. We present evidence that α1-PI forms a dimer sensitive to thiol reduction, suggesting cysteinyl-mediated dimerization of α1-PI. The α1-PI also demonstrated two types of inter-protein disulfide linkages: one resulting in homodimer and other involving heterodimer formation. TUG–Western immunoblot methodology was developed to identify the conformational changes in serpins. We found that the conformational changes in serpins by mixed-disulfide formation are due to unfolding and not to decomposition or degradation in TUG gels. Using fluorescence measurements with isolated tryptic fragments of fluorescence-labelled elastase, we observed that the cysteinyl232 in α1-PI interacted with the cysteinyl168 of elastase in the proteinase–inhibitor complex. Our data suggests that serpin thiols may play an important role in forming a stable serpin–proteinase complex.Key words: serpin, extracellular matrix, proteinase, reduction, oxidation, inhibitor.

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.

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