Chiral Dicopper Complexes with a Doubly Asymmetric Ligand as Models for the Tyrosinase Active Site: Synthesis, Structure, O2-Reactivity and Comparison with Their Symmetric Analogs

2009 ◽  
Vol 635 (8) ◽  
pp. 1123-1133 ◽  
Author(s):  
Ole Sander ◽  
Christian Näther ◽  
Felix Tuczek
Keyword(s):  
Active Site ◽  
Dicopper Complexes

Thiomethyl Substituted Dicopper Complexes: Attempts to Reproduce the Asymmetry of the Active Site from Type 3 Copper Enzymes

2013 ◽  
Vol 639 (8-9) ◽  
pp. 1477-1482 ◽  
Author(s):  
Wassim Rammal ◽  
Katalin Selmeczi ◽  
Christian Philouze ◽  
Eric Saint-Aman ◽  
Jean-Louis Pierre ◽  
...  
Keyword(s):  
Active Site ◽  
Copper Enzymes ◽  
The Asymmetry ◽  
Type 3 ◽  
Dicopper Complexes

Locating Al in the DABCO catalyst before and after Al extraction

1990 ◽  
Vol 48 (4) ◽  
pp. 265-267
Author(s):  
Kathleen B. Reuter
Keyword(s):  
Active Site ◽  
Inverse Relationship ◽  
Transverse Direction ◽  
Reaction Rate ◽  
Acid Phosphate ◽  
Fracture Surfaces ◽  
Al Content ◽  
Before And After ◽  
Relationship Of

The reaction rate and efficiency of piperazine to 1,4-diazabicyclo-octane (DABCO) depends on the Si/Al ratio of the MFI topology catalysts. The Al was shown to be the active site, however, in the Si/Al range of 30-200 the reaction rate increases as the Si/Al ratio increases. The objective of this work was to determine the location and concentration of Al to explain this inverse relationship of Al content with reaction rate.Two silicalite catalysts in the form of 1/16 inch SiO2/Al2O3 bonded extrudates were examined: catalyst A with a Si/Al of 83; and catalyst B, the acid/phosphate Al extracted form of catalyst A, with a Si/Al of 175. Five extrudates from each catalyst were fractured in the transverse direction and particles were obtained from the fracture surfaces near the center of the extrudate diameter. Particles were also obtained from the outside surfaces of five extrudates.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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.

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA

1990 ◽  
Vol 64 (01) ◽  
pp. 061-068 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoet ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Active Site ◽  
Peptide Bond ◽  
Tissue Type ◽  
Wild Type ◽  
Single Chain ◽  
Plasmin Activity ◽  
Soluble Fibrin ◽  
Type Plasminogen Activator ◽  
Presence And Absence ◽  
Chain Form

SummaryThe activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenaed to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA -Glul275, a recombinant single chain t-PA in which the Arg of the plasmin sensitiv e Arg275- Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s−1) per 1 μM enzyme. In the absence of fibrin stimulation, the vs for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s−1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s−1). In the presence of 1 μM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and, 33 pM s-1 respectively), whereas with 1 pM CNBr-digested fibrinogen, the vs for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s−1 respectively). In contrast, the vs for nPlg and rPlg-Ala740 by single chain rt-PA- G1u275, two-chain rt-PA-G1u275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.These findings confirm and establish: 1) that single chain t-PA is an active enzyme both in the presence and absence of fibrin stimulator; 2) that, in a system devoid of plasmin activity (rPlg- Ala740), the two-chain form of t-PA is about L5 times more active than the single chain form in the absence of fibrin but equipotent in the presence of desAAfibrin; and 3) that the mechanism of stimulation of plasminogen activation with single chain t-PA by CNBr-digested fibrinogen is different from that by soluble fibrin.

An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

1992 ◽  
Vol 67 (01) ◽  
pp. 095-100 ◽  
Author(s):  
Paul J Declerck ◽  
Leen Van Keer ◽  
Maria Verstreken ◽  
Désiré Collen
Keyword(s):  
Serine Protease ◽  
Plasminogen Activator ◽  
Active Site ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Protease Domain ◽  
Serine Protease Domain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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