(R)-3-Amidinophenylalanine-Derived Inhibitors of Factor Xa with a Novel Active-Site Binding Mode

2002 ◽  
Vol 383 (7-8) ◽  
Author(s):  
M. M. Mueller ◽  
S. Sperl ◽  
J. Stürzebecher ◽  
W. Bode ◽  
L. Moroder
Keyword(s):  
Active Site ◽  
Factor Xa ◽  
Binding Mode ◽  
Site Binding

Complexed and ligand-free high-resolution structures of urate oxidase (Uox) fromAspergillus flavus: a reassignment of the active-site binding mode

2004 ◽  
Vol 60 (3) ◽  
pp. 453-462 ◽  
Author(s):  
Pascal Retailleau ◽  
Nathalie Colloc'h ◽  
Denis Vivarès ◽  
Françoise Bonneté ◽  
Bertrand Castro ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Binding Mode ◽  
Urate Oxidase ◽  
Ligand Free ◽  
Site Binding

Ordered Cleavage of Prothrombin by Prothrombinase Results from Constrained Binding and Preferential Active Site Engagement by One of the Two Cleavage Sites in Prothrombin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 335-335 ◽  
Author(s):  
Ayse Hacisalihoglu ◽  
Sriram Krishnaswamy
Keyword(s):  
Active Site ◽  
Factor Xa ◽  
Substrate Affinity ◽  
Primary Role ◽  
Cleavage Sites ◽  
Proteolytic Activation ◽  
Active Site Probe ◽  
A Minor ◽  
Differential Action ◽  
Site Binding

Abstract The conversion of prothrombin to thrombin is a paradigm for proteolytic activation reactions wherein product is produced following cleavage at more than one site in the substrate. Human prothrombinase catalyzes thrombin formation by sequential cleavage of prothrombin at Arg320 followed by cleavage at Arg271. The largely ordered activation arises because Arg320 in intact prothrombin is hydrolyzed by prothrombinase with a Vmax that is ~30-fold greater than that for cleavage at Arg271 while substrate affinity is unchanged. Paradoxically, this phenomenon has been proposed to arise from the constrained binding of prothrombin to prothrombinase. The alternate proposal is that two interconverting forms of the enzyme differentially recognize and cleave the two sites in prothrombin. We have investigated substrate binding using prothrombinase assembled with a catalytically inactive recombinant factor Xa (XaS195A) in which Ser195 was replaced with Ala and a series of recombinant variants of prothrombin. The prothrombin variants included wild type prothrombin (IIWT) containing both cleavage sites, IIQ271 with Arg271 replaced with Gln and a single cleavable site at Arg320, IIQ320 with Arg320 replaced with Gln and a single cleavable site at Arg271 and the uncleavable IIQQ containing both Gln substitutions. Titration of XaS195A assembled into prothrombinase with increasing concentrations of the fluorescent active site probe p-aminobenzamidine (PAB) produced a saturable increase in fluorescence confirming the expected ability of XaS195A to bind ligands, such as PAB, at the active site despite its lack of catalytic activity. The addition of a saturating concentration of IIWT to reaction mixtures containing PAB and prothrombinase assembled with XaS195A resulted in a decrease in PAB fluorescence arising from the engagement of the active site by the substrate and the associated displacement of PAB. Only a minor change in PAB fluorescence was observed with IIQQ even though this uncleavable derivative binds to prothrombinase with the same affinity as IIWT. Thus, features of the substrate-enzyme interaction that determine affinity are independent of active site engagement by the substrate. Saturating concentrations of IIQ271 displaced PAB from the active site of XaS195A within prothrombinase to the same extent observed with IIWT while essentially no fluorescence decrease was observed with IIQ320. It follows that although all prothrombin derivatives bind with similar affinity to prothrombinase, elements flanking Arg320 can readily engage the active site of XaS195A within the enzyme complex while those flanking the Arg271 site cannot. Our equilibrium binding measurements establish a primary role for active site binding in determining the perceived rate constant for catalysis without influencing substrate affinity. The differential action of prothrombinase on the two cleavage sites in prothrombin likely arises from constraints imposed by active site-independent interactions that drive substrate affinity and permit active site docking by the Arg320 site in the substrate but not the Arg271 site. These constraints in active site engagement explain asymmetry in the recognition of the two bonds in intact prothrombin and its ordered cleavage by prothrombinase.

Protein modeling and active site binding mode interactions of myrosinase–sinigrin in Brassica juncea—An in silico approach

2011 ◽  
Vol 29 (5) ◽  
pp. 740-746 ◽  
Author(s):  
Rajender Kumar ◽  
Sandeep Kumar ◽  
Seema Sangwan ◽  
Inderjit Singh Yadav ◽  
Rakesh Yadav
Keyword(s):  
Brassica Juncea ◽  
Active Site ◽  
In Silico ◽  
Binding Mode ◽  
Protein Modeling ◽  
Mode Interactions ◽  
Site Binding

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

1986 ◽  
Vol 56 (03) ◽  
pp. 349-352 ◽  
Author(s):  
A Tripodi ◽  
A Krachmalnicoff ◽  
P M Mannucci
Keyword(s):  
Venous Thromboembolism ◽  
Active Site ◽  
Inhibitory Activity ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Defect ◽  
Affinity Adsorption ◽  
Italian Family ◽  
Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

Recent insights into lytic polysaccharide monooxygenases (LPMOs)

2018 ◽  
Vol 46 (6) ◽  
pp. 1431-1447 ◽  
Author(s):  
Tobias Tandrup ◽  
Kristian E. H. Frandsen ◽  
Katja S. Johansen ◽  
Jean-Guy Berrin ◽  
Leila Lo Leggio
Keyword(s):  
Active Site ◽  
Room Temperature ◽  
Experimental Studies ◽  
Binding Mode ◽  
Structural Features ◽  
Oxygen Species ◽  
Animal Kingdom ◽  
X Ray ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

New hypotheses for the binding mode of 4- and 7-substituted indazoles in the active site of neuronal nitric oxide synthase

2012 ◽  
Vol 20 (17) ◽  
pp. 5296-5304 ◽  
Author(s):  
Elodie Lohou ◽  
Jana Sopkova-de Oliveira Santos ◽  
Pascale Schumann-Bard ◽  
Michel Boulouard ◽  
Silvia Stiebing ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Active Site ◽  
Neuronal Nitric Oxide Synthase ◽  
Binding Mode ◽  
Oxide Synthase
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