scholarly journals Structural aspects of the effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase

1999 ◽  
Vol 337 (3) ◽  
pp. 373-377 ◽  
Author(s):  
Ruth GORDON-WEEKS ◽  
Saroj PARMAR ◽  
T. G. Emyr DAVIES ◽  
Roger A. LEIGH
Keyword(s):  
Nitrogen Atom ◽  
Active Site ◽  
General Structure ◽  
Specific Inhibitor ◽  
Carbon Chain ◽  
Proton Pumping ◽  
Azido Group ◽  
Higher Plant ◽  
Plant Vacuoles ◽  
Increased Activity

The bisphosphonates (general structure PO3-R-PO3) competitively inhibit soluble and membrane-bound inorganic pyrophosphatases (PPases) with differing degrees of specificity. Aminomethylenebisphosphonate (AMBP; HC(PO3)2NH2) is a potent, specific inhibitor of the PPase of higher plant vacuoles (V-PPase). To explore the possibility of constructing photoactivatable probes from bisphosphonates to label the active site of V-PPase we analysed the effects of different analogues on the hydrolytic and proton pumping activity of the enzyme. Bisphosphonates with a range of structures inhibited competitively and the effects on PPi hydrolysis correlated with the effects on proton pumping. Low-molecular-mass bisphosphonates containing hydrophilic groups (α-NH2 or OH) were the most effective, suggesting that the catalytic site is in a restricted polar pocket. Bisphosphonates containing a benzene ring were less active but the introduction of a nitrogen atom into the ring increased activity. Compounds of the general formula NH2(CH2)nC(PO3)2OH were more inhibitory than compounds of the H(CH2)nC(PO3)2NH2, NH2(CH2)nC(PO3)2NH2 or OH(CH2)nC(PO3)2NH2 series, with activity decreasing as n increased. A nitrogen atom in the carbon chain increased activity but activity was decreased by the presence of an oxygen atom. An analogue with a ring attached via a four-carbon chain, which included an amide linkage and a hydroxy group on the α-carbon atom, inhibited competitively (Ki = 62.0 µM), suggesting that it may be possible to design bisphosphonate inhibitors which contain a photoactivatable azido group for photoaffinity labelling of V-PPase active site.

Methods for the Isolation of Higher Plant Vacuoles

1988 ◽  
pp. 180-203
Author(s):  
A. Pugin ◽  
A. Kurkdjian ◽  
D. Le Quôc ◽  
K. Le Quôc
Keyword(s):  
Higher Plant ◽  
Plant Vacuoles

Proton pumping pyrophosphatase from higher plant mitochondria

1999 ◽  
Vol 105 (4) ◽  
pp. 763-768 ◽  
Author(s):  
Angelo Vianello ◽  
Francesco Macrì
Keyword(s):  
Plant Mitochondria ◽  
Proton Pumping ◽  
Higher Plant

scholarly journals Efficient co-expression of a recombinant staphopain A and its inhibitor staphostatin A in Escherichia coli

2004 ◽  
Vol 385 (1) ◽  
pp. 181-187 ◽  
Author(s):  
Benedykt WLADYKA ◽  
Katarzyna PUZIA ◽  
Adam DUBIN
Keyword(s):  
Active Site ◽  
Genomic Dna ◽  
Specific Inhibitor ◽  
Insoluble Fraction ◽  
Immobilized Metal Affinity Chromatography ◽  
Expression Vectors ◽  
Native Conformation ◽  
Natural Sources ◽  
Metal Affinity ◽  
Genes Encoding

Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni2+-nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.

scholarly journals Phosphonate monoester inhibitors of class A β-lactamases

1991 ◽  
Vol 275 (3) ◽  
pp. 793-795 ◽  
Author(s):  
J Rahil ◽  
R F Pratt
Keyword(s):  
Enzyme Inhibition ◽  
Mechanism Of Action ◽  
Active Site ◽  
General Structure ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Inhibitory Ability ◽  
Class A ◽  
Structure Formula ◽  
Slow Turnover

Phosphonate monoesters with the general structure: [formula: see text] are inhibitors of representative class A and class C beta-lactamases. This result extends the range of this type of inhibitor to the class A enzymes. Compounds where X is an electron-withdrawing substituent are better inhibitors than the unsubstituted analogue (X = H), and enzyme inhibition is concerted with stoichiometric release of the substituted phenol. Slow turnover of the phosphonates also occurs. These observations support the proposition that the mechanism of action of these inhibitors involves phosphorylation of the beta-lactamase active site. The inhibitory ability of these phosphonates suggests that the beta-lactamase active site is very effective at stabilizing negatively charged transition states. One of the compounds described also inactivated the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase/transpeptidase.

Structural analysis of mycobacterial branched-chain aminotransferase: implications for inhibitor design

2010 ◽  
Vol 66 (5) ◽  
pp. 549-557 ◽  
Author(s):  
Alina Castell ◽  
Christian Mille ◽  
Torsten Unge
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Specific Inhibitor ◽  
Inhibition Assay ◽  
Active Site Residues ◽  
X Ray ◽  
Sequence Identity ◽  
Branched Chain ◽  
Functional Analyses ◽  
Branched Chain Aminotransferase

The branched-chain aminotransferase (BCAT) ofMycobacterium tuberculosishas been characterized as being essential to the survival of the bacterium. The enzyme is pyridoxal 5′-phosphate-dependent and belongs to the aminotransferase IIIa subfamily, to which the human BCATs also belong. The overall sequence similarity is high within the subfamily and the sequence identity among the active-site residues is high. In order to identify structurally unique features ofM. tuberculosisBCAT, X-ray structural and functional analyses of the closely related BCAT fromM. smegmatiswere carried out. The crystal structures include the apo form at 2.2 Å resolution and a 1.9 Å structure of the holo form cocrystallized with the inhibitorO-benzylhydroxylamine (Obe). The analyses highlighted the active-site residues Tyr209 and Gly243 as being structurally unique characteristics of the mycobacterial BCATs relative to the human BCATs. The inhibitory activities of Obe and ammonium sulfate were verified in an inhibition assay. Modelling of the inhibitor Obe in the substrate pocket indicated potential for the design of a mycobacterial-specific inhibitor.

Structures of human cytosolic and mitochondrial nucleotidases: implications for structure-based design of selective inhibitors

2014 ◽  
Vol 70 (2) ◽  
pp. 461-470 ◽  
Author(s):  
Petr Pachl ◽  
Milan Fábry ◽  
Ivan Rosenberg ◽  
Ondřej Šimák ◽  
Pavlína Řezáčová ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Specific Inhibitor ◽  
Detailed Comparison ◽  
Atomic Resolution ◽  
Phosphate Anion ◽  
Protonation State ◽  
Interatomic Distances ◽  
Enzyme Active Site ◽  
Resolution Structure

The human 5′(3′)-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.

scholarly journals Mutations in a Conserved Residue in the Influenza Virus Neuraminidase Active Site Decreases Sensitivity to Neu5Ac2en-Derived Inhibitors

1998 ◽  
Vol 72 (3) ◽  
pp. 2456-2462 ◽  
Author(s):  
Jennifer L. McKimm-Breschkin ◽  
Anjali Sahasrabudhe ◽  
Tony J. Blick ◽  
Mandy McDonald ◽  
Peter M. Colman ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Active Site ◽  
Liquid Culture ◽  
Specific Inhibitor ◽  
Side Chain ◽  
Enzyme Assays ◽  
Hydrophobic Group ◽  
Influenza Virus Neuraminidase ◽  
Conserved Residue

ABSTRACT The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.

Neutralization of Human Extrinsic (Tissue-Type) Plasminogen Activator in Human Plasma: No Evidence for a Specific Inhibitor

1981 ◽  
Vol 46 (03) ◽  
pp. 662-665 ◽  
Author(s):  
C Korninger ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Rate Constant ◽  
Active Site ◽  
Gel Filtration ◽  
Specific Inhibitor ◽  
Culture Fluid ◽  
Void Volume ◽  
Tissue Type ◽  
Α2 Macroglobulin

SummaryHuman extrinsic plasminogen activator (EPA), highly purified from a melanoma cell culture fluid is inactivated in human plasma with a half-life (t ½) of 90–105 min. Gel filtration on Ultrogel AcA 34 of mixtures of 125I-labeled EPA and human plasma, incubated at 37°C, revealed the progressive formation of two radioactive components, one with an apparent Mr of 150,000 and one eluting at the void volume. The component with an Mr of 150,000 was identified as consisting at least in part of EPA-α2-antiplasmin complex since: 1) it reacted with antibodies against α2-antiplasmin, but not with antibodies against the other known plasma protease inhibitors, and 2) formation of this component was strongly reduced in plasma specifically depleted in α2-antiplasmin or when the active site of EPA was blocked. The component eluting at the void volume was identified as consisting at least in part of EPA-α2-macroglobulin complex since: 1) it only reacted with antibodies against these two proteins and 2) was not formed in plasma depleted in α2-macroglobulin or when the active site of EPA was blocked.In purified systems α2-antiplasmin inhibited one-chain EPA with a rate constant of 60 M-1s-1 and two-chain EPA with a rate constant of 130 M-1s-1, which corresponds to a t ½ in plasma of 180 min or 90 min, respectively. α2-Macroglobulin inhibited one-chain EPA with a rate constant of 15 M-1s-1 and two-chain EPA with a rate constant of 30 M-1s-1, which corresponds to a t ½ plasma of 4 or 2 hrs.All these findings taken together indicate that EPA is slowly neutralized in human plasma primarily by α2-antiplasmin and to a lesser extent by α2-macroglobulin. There appears to be no specific inhibitor in human plasma, which would inactivate EPA either rapidly or to a significant extent.

Effects of Plasma Kallikrein Specific Inhibitor and Active-site Blocked Factor VIIa on the Pulmonary Vascular Injury Induced by Endotoxin in Rats

1997 ◽  
Vol 78 (04) ◽  
pp. 1209-1214 ◽  
Author(s):  
Mitsuhiro Uchiba ◽  
Kenji Okajima ◽  
Kazunori Murakami ◽  
Hiroaki Okabe ◽  
Shosuke Okamoto ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Active Site ◽  
Factor Viia ◽  
Specific Inhibitor ◽  
Coagulation System ◽  
Plasma Kallikrein ◽  
Extrinsic Pathway ◽  
E Coli ◽  
Intravascular Coagulation

SummaryThe acute respiratory distress syndrome (ARDS) is a serious complication of sepsis. To evaluate the role of the coagulation system in the pathogenesis of ARDS in sepsis, we examined the effects of the administration of a synthetic plasma kallikrein specific inhibitor (PKSI) and of active-site blocked factor VIIa (DEGR-VIIa) on the pulmonary vascular injury induced by E. coli endotoxin (ET) in rats. Administration of PKSI prevented the pulmonary vascular injury induced by ET as well as pulmonary histological changes in animals administered ET, but it did not affect the intravascular coagulation. The opposite effect was seen with DEGR-VIIa, which prevented the intravascular coagulation but not the pulmonary vascular injury. PKSI did not inhibit the activation of the complement system induced by ET leading to the activation of neutrophils.Findings suggest that PKSI may prevent the pulmonary vascular injury induced by ET by inhibiting kallikrein, which activates the neutrophils. The intrinsic pathway of coagulation may be more important than the extrinsic pathway in the pulmonary vascular injury produced byET.

scholarly journals Theoretical Studies on the Mechanism of the Azido-Tetrazole of Azido-s-triazine

2015 ◽  
Vol 10 (2) ◽  
pp. 1934578X1501000
Author(s):  
Xiao-Qin Liang ◽  
Jin-Jun Zhou ◽  
Yan Zheng ◽  
Feng Ma
Keyword(s):  
Nitrogen Atom ◽  
Electronic Structures ◽  
Bond Angle ◽  
Nbo Analysis ◽  
Molecular Structures ◽  
Azido Group ◽  
Free Energy Barrier ◽  
Azide Group ◽  
Cyclization Process ◽  
Terminal Nitrogen Atom

The B3LYP/aug-cc-pvDZ level of theory has been applied to the study of the molecular structures, electronic structures and the azido-tetrazole isomerization of 1-azido-s-triazine, 1,3-diazido-s-triazine and 1,3,5-triazido-s-triazine. NBO analysis was applied to investigate the atomic natural charge and stabilization interaction energies among molecules. The results showed that the reaction initially proceeds through the loss of the linearity of the azido group and the approaching of the terminal nitrogen atom of the azide group to the nitrogen atom of the ring. This is followed by an attack of the lone pairs on N atoms in the ring to the azido group, leading to the formation of the N-N bonds. Many factors, including bending of the bond angle, electrostatic attraction, orbital delocalization and the stabilization interaction give rise to a large free energy barrier for the cyclization process. The results also show that the second and third cyclization is relatively easier than the first one.

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