Academic literature on the topic 'Substrate enzyme complex'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the lists of relevant articles, books, theses, conference reports, and other scholarly sources on the topic 'Substrate enzyme complex.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Journal articles on the topic "Substrate enzyme complex"
1
Christianson, David W., and William N. Lipscomb. "Carboxypeptidase A: novel enzyme-substrate-product complex." Journal of the American Chemical Society 109, no. 18 (September 1987): 5536–38. http://dx.doi.org/10.1021/ja00252a046.
Full text
2
Athipornchai, Anan, Nattisa Niyomtham, Wachirachai Pabuprapap, Vachiraporn Ajavakom, Maria Duca, Stéphane Azoulay, and Apichart Suksamrarn. "Potent Tyrosinase Inhibitory Activity of Curcuminoid Analogues and Inhibition Kinetics Studies." Cosmetics 8, no. 2 (May 4, 2021): 35. http://dx.doi.org/10.3390/cosmetics8020035.
Full textAbstract:
Natural tyrosinase inhibitors from herbal plants are promising therapeutic agents for skincare and cosmetic products. Natural curcuminoids exhibit weak antityrosinase properties. The structural modification of curcumin, the major curcuminoid from Curcuma longa, gave 14 analogues. The tyrosinase inhibitory activity of the natural curcuminoids and the modified analogues on both L-tyrosine and DOPA substrates were evaluated. The inhibition kinetics were also undertaken. For analogues with potent activity on the L-tyrosine substrate, the isoxazole analogue 12 and two reduced analogues, hexahydrocurcumin (16) and the α,β-unsaturated analogue 17, showed IC50 values of 8.3, 14.6 and 9.4 µM, and were 20.9-, 11.9- and 18.4-fold more active, respectively, than kojic acid, the reference compound. For the analogues with potent antityrosinase on DOPA substrate, the dimethylated analogue 5 exhibited the strongest antityrosinase activity against the DOPA substrate, with the IC50 value of 8.0 µM, which was 16.6-fold more active than kojic acid. The inhibition kinetics revealed that curcuminoid 5 could bind with both free enzyme and with the enzyme–substrate complex. It acted as a competitive–uncompetitive mixed-II type inhibitor. Curcuminoid 17 could bind with both free enzyme and the enzyme–substrate complex. The results indicated that 17 acted as a competitive–uncompetitive mixed-I type inhibitor, while curcuminoid 12 was a noncompetitive inhibitor which bound with both free enzymes and the enzyme–substrate complex. These potent analogues might serve as new potential tyrosinase inhibitors for the prevention and treatment of skin pigmentation disorders.
3
Sulaiman, Noorul Aini. "Combining Docking and Molecular Dynamic of Protease from Bacillus lehensis G1." Journal of Engineering and Science Research 2, no. 1 (February 28, 2018): 1–5. http://dx.doi.org/10.26666/rmp.jesr.2018.1.1.
Full textAbstract:
Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use.
4
Xiang, Yong, Khanita Karaveg, and Kelley W. Moremen. "Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway." Proceedings of the National Academy of Sciences 113, no. 49 (November 17, 2016): E7890—E7899. http://dx.doi.org/10.1073/pnas.1611213113.
Full textAbstract:
Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-mannosidases, including endoplasmic reticulum (ER) α-mannosidase I (ERManI) and Golgi α-mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca2+ ion with a lanthanum (La3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La3+-bound enzymes with Man9GlcNAc2 substrate analogs revealed enzyme–substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.
5
Traving, Sachia J., Uffe H. Thygesen, Lasse Riemann, and Colin A. Stedmon. "A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?" Applied and Environmental Microbiology 81, no. 21 (August 7, 2015): 7385–93. http://dx.doi.org/10.1128/aem.02070-15.
Full textAbstract:
ABSTRACTAn initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration.
6
AHMAD, ABDUL AZIZ, Hamzah Mohd Salleh, and IBRAHIM ALI NOORBATCHA. "ROLE OF SUBSTRATE BINDING ON THE PROTEIN DYNAMICS OF AN ENDOGLUCANASE FROM FUSARIUM OXYSPORUM AT DIFFERENT TEMPERATURES." IIUM Engineering Journal 19, no. 1 (June 5, 2018): 307–14. http://dx.doi.org/10.31436/iiumej.v19i1.894.
Full textAbstract:
: Thermostability is an important requirement for protein function, and one goal of protein engineering is improvement of activity of the enzymes at higher temperatures, particularly for industrial applications. Computational approaches to investigate factors influencing thermostability of proteins are becoming researchers’ choice. This study investigates the influence of substrate binding on the protein dynamics by comparing the molecular dynamics simulations of substrate-enzyme complex against un-bound enzyme, using endoglucanase I from Fusarium oxysporum. Endoglucanase-substrate complex was prepared by docking and molecular dynamics simulations were carried out at three different temperatures, 313 K, 333 K and 353 K. Our finding shows that the secondary structures for substrate-enzyme complex show more fluctuations relative to un-complexed structure. The same trend was observed for solvent accessible surface area and radius of gyration. At the highest temperature studied (353 K), the substrate-enzyme complex form showed the highest fluctuations. The fluctuations around the active site regions reach a minimum at the optimum temperature, compared to the other structural regions and other temperatures.
ABSTRAK: Kestabilan (ketahanan) terhadap haba merupakan keperluan yang penting untuk fungsi protin, salah satu matlamat kejuruteraan protin adalah penambahbaikan aktiviti enzim pada suhu yang tinggi khususnya untuk aplikasi industri. Kini para penyelidik memilih kaedah komputasi, bagi mengkaji faktor yang mempengaruhi kestabilan terhadap haba. Kajian ini menyelidik pengaruh ikatan substrat pada protin dengan membandingkan simulasi molekular dinamik diantara substrat-enzim kompleks dan enzim sahaja, menggunakan endoglucanase I dari Fusarium oxysporum. Kompleks endoglucanase-substrat disediakan melalui kaedah docking dan simulasi molekular dinamik dilakukan pada suhu 313 K, 333 K dan 353 K. Kajian kami menunjukkan struktur sekunder bagi substrat-enzim kompleks kurang stabil berbanding enzim sahaja. Pola yang sama bagi luas permukaan boleh dicapai pelarut (SASA) dan jejari gyrasi. Pada suhu tertinggi dikaji (353 K), substrat-enzim kompleks paling tidak stabil. Pada suhu optimum, kadar ubah-ubah sekitar amino asid aktif adalah minimum berbanding struktur dan suhu lain.
7
Vinnov, Aleksey, and Dmitro Prasol. "ENZYMATIC HYDROLYSIS KINETIC ANALYSIS OF THE VARIOUS SOURCE PROTEINS." Acta Universitatis Cibiniensis. Series E: Food Technology 17, no. 2 (December 1, 2013): 15–20. http://dx.doi.org/10.2478/aucft-2013-0007.
Full textAbstract:
Abstract The chemical composition marble goby and sunflower meal is presented. The experimental results of enzymatic hydrolysis velocity in dependence of the protein concentration in substrate systems are set. Michaelis constants values for industrial proteolytic enzymes Corolase ® L10 and Corolase ® L7089 are calculated. The application experimental - theoretical kinetic analysis for protease / proteins congeniality determine in complex dispersion substrate systems expediency was confirmed. It is determined that microbial enzyme drag Corolase ® L7089 has a higher congeniality to proteins of all the tested substrates than plant enzyme drag Corolase ® L10
8
Kim, Han-Woo, and Kazuhiko Ishikawa. "Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots." Biochemical Journal 437, no. 2 (June 28, 2011): 223–30. http://dx.doi.org/10.1042/bj20110292.
Full textAbstract:
A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65–2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite −1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites −1 and +1. Subsite −2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite −1. A rare enzyme–substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme–substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.
9
Freeman, C., and J. J. Hopwood. "Human α-l-iduronidase. Catalytic properties and an integrated role in the lysosomal degradation of heparan sulphate." Biochemical Journal 282, no. 3 (March 15, 1992): 899–908. http://dx.doi.org/10.1042/bj2820899.
Full textAbstract:
The kinetic parameters (Km and kcat) of human liver alpha-L-iduronidase were determined with a variety of heparin-derived disaccharide and tetrasaccharide substrates. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrates heparin and heparan sulphate were maintained, were hydrolysed with catalytic efficiencies up to 255 times that observed for the simplest disaccharide substrate to be hydrolysed. The major aglycone structure that influenced both substrate binding and enzyme activity was the presence of a C-6 sulphate ester on the residue adjacent to the iduronic acid residue being hydrolysed. Sulphate ions and a number of substrate and product analogues were potent inhibitors of enzyme activity. Human liver alpha-L-iduronidase activity towards 4-methylumbelliferyl alpha-L-iduronide at pH 4.8 had two Km values of 37 microM and 1.92 mM with corresponding kcat. values of 299 and 650 mol of product formed/min per mol of enzyme respectively, which may explain the wide range of Km values previously reported for alpha-L-iduronidase activity toward its substrate. Skin fibroblast alpha-L-iduronidase activity towards the heparin-derived oligosaccharides was influenced by the same substrate aglycone structural features as was observed for the human liver enzyme. A comparison was made of the effect of substrate aglycone structure upon catalytic activities of the enzymes which act to degrade the highly sulphated regions of heparan sulphate. A model was proposed whereby the substrate is directed from alpha-L-iduronidase to subsequent enzyme activities to ensure the efficient degradation of heparan sulphate.
10
Freeman, C., and J. J. Hopwood. "Human liver N-acetylglucosamine-6-sulphate sulphatase. Catalytic properties." Biochemical Journal 246, no. 2 (September 1, 1987): 355–65. http://dx.doi.org/10.1042/bj2460355.
Full textAbstract:
Kinetic parameters (Km and kcat.) of the two major forms (A and B) and a minor form (C) of human liver N-acetylglucosamine-6-sulphate sulphatase [Freeman, Clements & Hopwood (1987) Biochem. J. 246, 347-354] were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin, heparan sulphate and keratan sulphate. Enzyme activity is highly specific towards glucosamine 6-sulphate or glucose 6-sulphate residues. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are hydrolysed with catalytic efficiencies up to 3900 times above that observed for the monosaccharide substrate N-acetylglucosamine 6-sulphate. Forms A and B both desulphate substrates derived from keratan sulphate and heparin. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue 6-carboxy and 2-sulphate ester groups for heparin-derived substrates and penultimate-residue 6-sulphate ester groups for keratan sulphate-derived substrates. The 4-hydroxy group of the N-acetylglucosamine 6-sulphate or the 2-sulphaminoglucosamine 6-sulphate under enzymic attack is involved in the catalytic mechanism. The presence of a 2-amino group in place of a 2-acetamido or a 2-sulphoamino group considerably decreases the catalytic efficiency of the sulphatase, particularly in the absence of a penultimate-aglycone-residue 6-carboxy group. Both forms A and B are exo-enzymes, since activity towards internal sulphate ester bonds was not observed. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone 2-sulphate ester, 6-carboxy group and 6-sulphate ester groups on the glucosamine 6-sulphate residue under attack considerably affects the pH response. Sulphate and phosphate ions are potent inhibitors of enzyme activity.
Dissertations / Theses on the topic "Substrate enzyme complex"
1
Pauthe, Emmanuel. "Approches cinétiques et moléculaires de la reconnaissance enzyme-substrat : application à l'étude de l'activité protéolytique de la thermolysine." Compiègne, 1998. http://www.theses.fr/1998COMP1139.
Full textAbstract:
L’accomplissement de tout acte protéolytique implique nécessairement la formation d'un complexe entre l'enzyme et son substrat. Par différentes approches cinétiques, spectroscopiques et moléculaires nous avons cherché à caractériser les phénomènes mis en jeu au cours de l'hydrolyse, par la thermolysine, de petits peptides en milieu biomimétiques. Cette étude a été conduite à l'interface entre la biochimie, la biophysique, la chimie et la physique. Dans un premier temps, nous nous sommes intéressés au comportement catalytique de la thermolysine sur des substrats modèles et en milieu modifié. Nous avons montré d'une part, que l'ajout d'additifs polyhydroxylés influence grandement l'activité de la thermolysine et d'autre part, affine les connaissances sur la spécificité et la sélectivité de cette enzyme (en particulier, mise en évidence de l'influence du résidu P'2 dans le mécanisme). Dans un deuxième temps, nous présentons des études structurales des peptides substrats en milieu modifié. Nous avons mis en évidence l'absence d'influence du micro-environnement contenant une forte proportion de glycérol sur la conformation des molécules de substrat et le rôle possible de leur structure tridimensionnelle quant à leur hydrolyse. Ces études ont été étendues à un autre modèle peptidique, de forme cyclique ou linéaire, et corrélées aux résultats cinétiques. Dans un troisième temps, par deux approches différentes, nous avons abordé l'étude des relations structure-fonction de la thermolysine. Expérimentalement, par des études cinétiques avec l'enzyme immobilisée et des déterminations de sa structure par spectroscopie laser Raman, nous montrons que l'enzyme est très peu sensible au micro-environnement. Théoriquement en analysant, par modélisation, l'interaction de la thermolysine avec un tripeptide substrat, nous avons mis en évidence des changements de conformation du substrat et/ou des mouvements du site actif enzymatique au cours de l'acte catalytique.
2
Borel, Franck. "Contributions à l'étude des interactions entre les ARNtser et la séryl-ARNt synthétase d'Escherichia coli et de Saccharomyces cerevisiae." Université Joseph Fourier (Grenoble), 1994. http://www.theses.fr/1994GRE10142.
Full textAbstract:
Ce travail est consacre a l'etude des mecanismes responsables de la reconnaissance entre la seryl-arnt synthetase d'escherichia coli et son substrat macromoleculaire, l'arnt#s#e#r. La premiere etape de ces travaux a consiste a surexprimer l'arnt#s#e#r#2 et l'arnt#s#e#r ambre suppresseur d'e. Coli. Pour cela, les genes synthetiques des deux arnt#s#e#r ont ete construits par assemblage de sept oligonucleotides chevauchants. L'utilisation d'un plasmide dont le nombre de copies est regule par la temperature permet d'obtenir des cellules bacteriennes contenant un taux d'arnt#s#e#r vingt fois superieur a celui present dans des cellules depourvues de plasmide. Grace a cette surexpression, un protocole de purification comportant deux etapes chromatographiques a pu etre developpe. Les arnt purifies ont ensuite ete utilises pour determiner la contribution du domaine n-terminal de la seryl-arnt synthetase d'e. Coli, a l'efficacite et a la specificite de la reaction d'aminoacylation. Les resultats obtenus, montrent que le domaine n-terminal depourvu de role catalytique lors de la reaction d'activation de l'acide amine, est indispensable, d'une part, au maintien d'une efficacite de catalyse elevee lors du transfert de l'acide amine active sur l'arnt et, d'autre part, a la specificite de reconnaissance des arnt#s#e#r. Parallelement, la mesure des constantes de dissociation de l'arnt#s#e#r#2, revele que la fixation de deux molecules d'arnt sur la seryl-arnt synthetase est regit par un mecanisme de type cooperatif. La derniere partie de ce travail montre que la seryl-arnt synthetase de saccharomyces cerevisiae ne peut etre surexprimee chez e. Coli. Le contexte bacterien ne permettant pas a la seryl-arnt synthetase de s. Cerevisiae d'acquerir une structure tridimensionnelle homogene. Les etudes menees, en parallele sur la seryl-arnt synthetase de s. Cerevisiae surexprimee dans son contexte naturel, revelent une repartition de cette enzyme en deux populations distinctes, et une mobilite electrophoretique, sur sds-page, plus elevee que celle obtenue pour l'enzyme surexprimee chez e. Coli
3
Lorber, Bernard. "Contribution a l'etude du systeme aspartyl-trna synthetase-trna**(asp) chez la levure saccharomyces cerevisiae." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13049.
Full text
4
Lengqvist, Johan. "Native protein mass spectrometry of nuclear receptor-ligand and enzyme-substrate complexes /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-116-4/.
Full text
5
Schroeder, Ewald. "Structural studies of #mu#-calpain, a novel calpain substrate, and a papain-leupeptin complex." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386677.
Full text
6
Thoresen, Mariska. "An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1017915.
Full text
7
Badruna, Louise. "Création d'enzymes multimodulaires à façon dédiées à la dégradation de substrats complexes." Thesis, Toulouse, INSA, 2017. http://www.theses.fr/2017ISAT0021/document.
Full textAbstract:
Pour réduire notre empreinte carbone sur l’environnement, il est urgent de développer des procédés industriels utilisant une source de carbone renouvelable comme la biomasse lignocellulosique. La paroi cellulaire végétale est un enchevêtrement complexe de cellulose, d’hémicelluloses et de lignines. Elle résiste aux attaques biologiques et chimiques mais limite le développement d’une bioéconomie responsable. Dans la Nature, des enzymes multimodulaires produites par certains microorganismes peuvent la déconstruire. Toutefois, ces enzymes sont majoritairement étudiées sur des substrats artificiels ou purifiés. Dans cette thèse, nous proposons de les étudier sur des substrats broyés puis des coupes de paille de blé brutes. Les enzymes multimodulaires sont préparées à façon en utilisant la propriété d’association covalente des protéines Jo et In. Nous avons ainsi associé la xylanase NpXyn11A de N. patriciarum avec deux modules non catalytiques : le CBM3a de C. thermocellum ou le CBM2b1 C. fimi ciblant la cellulose ou les xylanes respectivement. Les propriétés biochimiques de ces protéines chimériques ont été comparées aux modules sauvages. L’activité enzymatique des protéines chimériques a ensuite été étudiée sur des substrats solubles, jusqu’à des substrats insolubles comme le son et la paille de blé, notamment par immunocytochimie. Ce travail a mis en évidence l’importance de la relation enzymes/substrats pour une caractérisation in muro d’activité enzymatique et une meilleure compréhension de la déconstruction de la biomasse végétaleIn order to reduce our carbon footprint on the environment, it is more than urgent to develop new industrial process using a renewable carbon source such as lignocellulosic biomass. Plant cell walls consist of a complex network of cellulose, hemicelluloses and lignins that cross-link with each other mainly via non-covalent bonds. It is thus hardly surprising that plant biomass is rather recalcitrant to chemical or biological degradation. In the present era marked by the desire to build a green bioeconomy, this recalcitrance remains a key point. In Nature, the plant-based organic carbon contained within plant cell walls is mainly recycled by the action of cellulolytic microorganisms, producing multimodular enzymes. However, these enzymes are mainly characterized on artificial or purified substrates. In this thesis, we proposed to study multimodular enzymes on raw substrates such as wheat straw sections. The studied multimodular enzymes were associated thanks to the use of two small proteins Jo and In. Thus, we associated the xylanase NpXyn11A from N. patriciarum with two non-catalytic modules: CBM3a from C. thermocellum or CBM2b1 from C. fimi targeting cellulose or xylans respectively. Biochemical properties of these chimeric proteins and wild-type modules have been compared. The enzymatic activity of chimeric proteins has been studied on soluble substrates and compared to the activity on insoluble substrates, mainly by immunocytochemistry. This work highlighted the importance of the relationship enzymes/substrates and its key role to better understand the biomass deconstruction in muro
8
Fatih, Mustapha. "Contribution à la connaissance du chemin réactionnel des Glycéraldéhyde-3-phosphate déshydrogénases phosphorylantes : structures cristallines de complexes ternaires (enzyme mutée + cofacteur + substrat)." Nancy 1, 2000. http://docnum.univ-lorraine.fr/public/SCD_T_2000_0074_FATIH.pdf.
Full textAbstract:
Ces travaux concernent l'étude cristallographique de complexes ternaires de la Glycéraldéhyde-3-phosphate déshydrogénase phosphorylante de B. Stearothermophilus. Cette enzyme catalyse la phosphorylation oxydative du Glycéraldéhyde-3-phosphate en présence d'un cofacteur, le NAD, et de phosphate inorganique. Pour fixer les groupements phosphates de ces deux substrats, l'enzyme possède deux sites de reconnaissance anionique, dénommés respectivement Pi et Ps. Cependant, la façon dont ces deux sites contribuent à la fixation des deux groupements phosphate reste encore soumise à controverse et différents modèles ont été proposés. Afin de tester la validité des ces modèles, il semblait essentiel de pouvoir disposer d'évidences structurales directes. Deux mutants ont été étudiés : le mutant C149A, inactif et le mutant C149S, très faiblement actif. Des cristaux de ces deux mutants ont été obtenus dans des conditions originales, exemptes de tout anion. Ces nouvelles conditions de cristallisation ont conduit à de nouveaux empilements cristallins. L'analyse des structures des complexes binaires met en évidence des sites Pi libres de tout anion. Par contre, les sites Ps sont occupés par un anion sulfate issu probablement de la purification. Ceci montre que le site Ps a une meilleure affinité que le site Pi vis-à-vis des anions. Ce résultat a été confirmé par l'analyse des acides aminés impliqués dans la formation des sites Pi et Ps et par le fait que le potentiel électrostatique local est plus important au niveau du site Ps qu'au niveau site Pi. Les complexes ternaires que nous avons obtenus constituent la première structure tridimensionnelle d'une GAPDH en complexe avec son substrat physiologique. L'analyse de ces complexes a permis d'apporter des informations quant aux interactions enzyme-substrat. Nous montrons sans ambigüité que dans les deux complexes obtenus, le groupement phosphate du G3P est localisé dans le site Ps. De même, les facteurs moléculaires responsables de la stéréosélectivité vis-à-vis du D-G3P ont pu être élucidés. Cependant, le débat reste ouvert concernant, d'une part, la contribution de la boucle 205-210 à la catalyse et d'autre part, la conséquence de la formation de la liaison covalente entre l'enzyme et le substrat sur le positionnement des intermédiaires réactionnels dans le site actif.
9
Moniot, Sébastien. "Caractérisation Structurale et Fonctionnelle de deux Enzymes de la Famille des Aldéhyde déshydrogénases : la Glycéraldéhyde-3-Phosphate Déshydrogénase de B. stearothermophilus et l'Erythrose-4-Phosphate Déshydrogénase d'E. coli. : structures cristallographiques d'intermédiaires réactionnels et de complexes enzyme-substrat." Thesis, Nancy 1, 2008. http://www.theses.fr/2008NAN10091/document.
Full textAbstract:
Si la GAPDH est une enzyme bien caractérisée sur le plan biochimique et structural, la contribution de ses deux sites de reconnaissance anionique lors de la catalyse reste incomprise et nécessitait la détermination des structures de l'intermédiaire thioacylenzyme et du complexe enzyme-produit. Ce manuscrit présente la mise au point des conditions de cristallisation et d'accumulation de l'intermédiaire thioacylenzyme (suivi par microspectrophotométrie) et du complexe enzyme-produit, la résolution et l'analyse des structures cristallographiques correspondantes ainsi que les implications pour le mécanisme catalytique. Les résultats obtenus mettent notamment en évidence un mouvement de "va-et-vient" du substrat au cours de la catalyse entre les deux sites de reconnaissance anionique qui est lié à l'étape d'échange du cofacteur. Bien que structuralement proche des GAPDH, l’E4PDH est une enzyme pour laquelle peu de données sont disponibles. De nombreuses questions restent donc posées concernant notamment les déterminants de l'affinité pour le cofacteur, la nature du site de reconnaissance anionique, ou encore le mécanisme d'activation de la molécule d'eau nécessaire à une étape d'hydrolyse efficace. Sont présentées dans ce manuscrit trois structures cristallographiques de l'E4PDH d'E. coli, sous forme apoenzyme, en présence de phosphate ou en complexe avec un analogue de cofacteur. L'analyse de ces structures a notamment permis de caractériser l'unique site de reconnaissance anionique de l'enzyme, de proposer des hypothèses quant à la faible affinité de l'enzyme pour le cofacteur, et d'identifier un candidat possible pour l'activation de la molécule d'eau hydrolytiqueEven if GAPDH is well characterized from a biochemical and structural point of view, the contribution of its two anion recognition sites to catalysis is still matter of debate. This work presents the crystallization, the strategy for the accumulation of the thioacylenzyme intermediate and for the obtaining of the enzyme-product complex, the resolution and analysis of the corresponding crystallographic structures and the implications in terms of reaction mechanism. The results mainly shed light on the existence of a flip-flop movement of the substrate between the two anion recognition sites during catalysis which is related to the cofactor exchange step. Although structurally related to GAPDHs, the E4PDH is an enzyme fairly less characterized. Many interrogations thus remain on the determinants of cofactor affinity, on the nature of the anion recognition site or on the mechanism of activation of the water molecule that is needed for an efficient hydrolysis step. This dissertation presents the resolution of three crystallographic structures of the E4PDH from E. coli, under the apoenzyme form, in the presence of phosphate or in complex with a cofactor analog. The analysis f these structures allows the characterization of the unique anion recognition site of this enzyme, to propose structural hypotheses to explain the low affinity of this enzyme for its cofactor and also to identify possible candidates for the activation of the nucleophilic water molecule
10
Leclaire, Jacques. "Oxydations catalysees par les cytochromes p-450 et les systemes metalloporphyriniques modeles : cas des alcenes monosubstitues et du leucotriene b::(4)." Paris 6, 1987. http://www.theses.fr/1987PA066473.
Full textAbstract:
La selectivite des oxydations catalysees par les monooxygenases a cytochrome p-450 tient compte de deux facteurs principaux: 1) le role de la chaine proteique bordant le site actif du cytochrome p450. 2) la chimioselectivite du complexe a oxygene actif implique dans la reaction. Oxydation du phenoxy-6 hexene 1 et du leucotriene b4 fait intervenir des familles de cytochromes p-450 tres differentes. L'utilisation de systemes metalloporphyriniques permet de faire, en partie, la difference entre les facteurs dus a la reactivite intriseque de l'entite a oxygene actif et ceux dus a l'environnement proteique. L'apoproteine est absente et la reconnaissance du substrat est reduite a son strict minimum puisqu'elle n'est plus excitee que par la metalloporphyrine. Mise au point de catalyseurs selectifs d'oxydation des olefine monosubstituees
Books on the topic "Substrate enzyme complex"
1
Liaw, Ean-Tun. Characterization of substrate-velocity relationships for the cellulase enzyme complex from Trichoderma viride. 1989.
Find full textBook chapters on the topic "Substrate enzyme complex"
1
Gooch, Jan W. "Enzyme-Substrate Complex." In Encyclopedic Dictionary of Polymers, 891. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-6247-8_13678.
Full text
2
Varfolomeev, Sergey, Bella Grigorenko, Sofya Lushchekina, Patrick Masson, Galina Mahaeva, and Alexander Nemuchin. "Human cholinesterases." In ORGANOPHOSPHORUS NEUROTOXINS, 69–126. ru: Publishing Center RIOR, 2020. http://dx.doi.org/10.29039/21_069-126.
Full textAbstract:
The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.
3
Varfolomeev, Sergey, Bella Grigorenko, Sofya Lushchekina, and Alexander Nemuchin. "Human cholinesterases." In Organophosphorous Neurotoxins, 63–120. ru: Publishing Center RIOR, 2020. http://dx.doi.org/10.29039/chapter_5e4132b5f22366.15634219.
Full textAbstract:
The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.
4
Mao, Youdong. "Structure, Dynamics and Function of the 26S Proteasome." In Subcellular Biochemistry, 1–151. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-58971-4_1.
Full textAbstract:
AbstractThe 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal “processor” for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.
5
Wood, B. J. B., B. L. Ghosh, and S. N. Sinha. "Solid Substrate Fermentation in the Production of Enzyme Complex for Improved Jute Processing." In ACS Symposium Series, 46–59. Washington, DC: American Chemical Society, 1996. http://dx.doi.org/10.1021/bk-1996-0647.ch004.
Full text
6
Antonov, Vladimir K. "Enzyme-Substrate Complexes." In Chemistry of Proteolysis, 281–318. Berlin, Heidelberg: Springer Berlin Heidelberg, 1993. http://dx.doi.org/10.1007/978-3-662-00979-6_7.
Full text
7
Taylor, Neil R., Andrew K. J. Chong, Michael S. Pegg, and Mark von Itzstein. "Molecular modelling studies on the enzyme—substrate complex sialidase-N-acetylneuraminic acid and the mechanism of catalysis." In Trends in QSAR and Molecular Modelling 92, 478–80. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-011-1472-1_125.
Full text
8
Tsuge, Hideaki, and Toshiharu Tsurumura. "Reaction Mechanism of Mono-ADP-Ribosyltransferase Based on Structures of the Complex of Enzyme and Substrate Protein." In Endogenous ADP-Ribosylation, 69–87. Cham: Springer International Publishing, 2014. http://dx.doi.org/10.1007/82_2014_415.
Full text
9
Fiedler, F., and H. Hinz. "Kinetics of Bond Cleavages at Kallidin Release by Tissue Kallikrein: Cleavage of Two Peptide Bonds in a Single Enzyme-Substrate Complex ?" In Recent Progress on Kinins, 82–88. Basel: Birkhäuser Basel, 1992. http://dx.doi.org/10.1007/978-3-0348-7321-5_11.
Full text
10
Yon-Kahn, Jeannine, and Guy Hervé. "Formation of Enzyme-Substrate Complexes." In Molecular and Cellular Enzymology, 347–75. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-01228-0_11.
Full textConference papers on the topic "Substrate enzyme complex"
1
Hideshima, Taketoshi. "Oscillatory Reaction of Enzyme Caused by Gradual Entry of Substrate." In SLOW DYNAMICS IN COMPLEX SYSTEMS: 3rd International Symposium on Slow Dynamics in Complex Systems. AIP, 2004. http://dx.doi.org/10.1063/1.1764156.
Full text
2
Vermeer, C., BA M. Soute, and MM W. Ulrich. "IN VITRO CARBOXYLATION OF EXOGENOUS PROTEIN SUBSTRATES BY VITAMIN K-DEPENDENT CARBOXYLASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643994.
Full textAbstract:
In vivo treatment of experimental animals with vitamin K-antagonists induces the accumulation of non-carboxylated coagulation factor precursors in the liver, where they are tightly bound to vitamin K-dependent carboxylase. If hepatic carboxylase is isolated from warfarin-treated animals, it is obtained therefore almost exclusively in the form of an enzyme/substrate complex. If carboxylase is prepared from non-treated animals, on the other hand, the resulting enzyme is predominantly substrate-free. Small substrates like F L E E L or decarboxylated osteocalcinare carboxylated equally well by both types of carboxylase, but protein substrates(Mr > 30 000) are recognized exclusively by substrate-free carboxylase.Initial attempts to purify carboxylasewere performed with livers from warfarin-treated cows as a starting material. Antibodies against the normal blood coagulation factors crossreact with the hepatic precursor proteins so that the enzyme/substrate complexes could be specifically extracted from detergent-solubilized microsomes by the substrate/antibody interaction. This procedure resulted ina substantial purification of carboxylase, but because its endogenous substrate remained firmly bound, even after it had been carboxylated in vitro, the enzyme system was not suitable for the carboxylation of protein substrates.Therefore a second strategy was developed by which substrate-free carboxylase (from normal livers) was partly purified by sequential extraction of the microsomal membranes with detergents, followed by ammonium sulfate precipitation and size exclusion chromatography.This procedure resulted in a soluble carboxylase complex, still consisting of 7 proteins and phosphatidylcholine. Although further dissociation of the complex resulted in a complete loss of activity, it is not sure if all components play a role in the carboxylation reaction. Exogenous substrates which could be carboxylated by substrate-free carboxylase were: the penta-peptide F L E E L, descarboxyprothrombin from bovine plasma, thermally decarboxylated osteocalcin from bovine bone and non-car-boxy lated coagulaton factor precursors which had been produced by recombinant-DNA techniques in various laboratories. The . efficiency of CO^ incorporation was: 1 mole per 100 moles of F L E E L, 1 mole per 240 moles of descarboxy-prothrombin, 1 mole per mole of decarboxylated osteocalcin and 8 moles per mole of a recombinant factor IX precursor. We assume that the high efficiency with which the recombinant coagulation factor precursors were carboxylated is due to the presence of at least part of their leader sequence. The importance of the aminoacid chain preceding the first carboxylatable Glu residue is demonstrated by the fact that descarboxylated osteocalcin of bovine origin is carboxylated with a relatively high efficiency, whereas descarboxylated osteocalcin from monkey bone is not recognized atal.. Yet the only difference between the two substrates is found in their aminoacids 3 and 4, whereas the first carboxylatable Glu occurs at position 17. It seems, therefore, that the aminoacids 1-16 in bovine osteocalcin mimic to some extent part of the leader sequence in the coagulation factor precursors. Chemical or biochemical modification of decarboxylated osteocalcin might reveal which structural features contribute to its recognition by hepatic carboxylase.The optimal conditions for carboxylation include a high concentration of dithiols (e.g. DTT) and under these conditions disulfide bridges are reduced. Obviously this will lead to a complete destruction of the biological activity of various carboxylated products. Therefore we have searched for a more natural reducing system and it was found that the bacterial thioredoxin/thiore-doxin-reductase system in the presence of 40 uM NADFH was able to replace DTT in the reaction mixtures. Since a comparable system also occurs in calf liver it seems not unlikely that this is the physiological counterpart of the dithiols used in vitro.
3
Hrmova, Maria, Joseph N. Varghese, Ross De Gori, Brian J. Smith, Hugues Driguez, Jon K. Fairweather, Andrea Vasella, and Geoffrey B. Fincher. "CATALYTIC MECHANISMS AND THE BASIS OF SUBSTRATE SPECIFICITY OF BETA-D-GLUCAN GLUCOHYDROLASES: CRYSTAL STRUCTURES OF THE ENZYME IN COMPLEX WITH GLUCOSIDE INHIBITORS AND SUBSTRATE ANALOGUES." In XXIst International Carbohydrate Symposium 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.467.
Full text
4
Verhamme, I. M. A., and G. W. K. van Dedem. "KINETICS OF HYDROLYSIS OF THE CHROMOGENIC SUBSTRATE S2238 BY ALPHA-THROMBIN : INFLUENCE OF THE pH AND THE IONIC STRENGTH." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643276.
Full textAbstract:
The knowledge of the pH and ionic strength dependence of kcat and Km for the hydrolysis of S2238 (HD-phe-pip-arg-pNA.2HC1) by alpha-thrombin is essential in determining optimal reaction conditions of residual enzyme in systems where also protease inhibitors and glycosaminoglycan catalysts play a role.We studied the kinetic behavior of S2238 in piperazine/glycylglycine/NaOH buffers from pH 6 to 11 and with a calculated ionic strength up to 0.7 M taking into account the pH-dependent concentration of the buffer species. The kinetic parameters of 60 Michaelis-Menten substrate functions were used for the setup of ionic strength and pH profiles. The kcat values are dependent upon the ionic strength, increasing steeply up to about 0.3 M and decreasing again at high ionic strength.The Km however,reflecting the affinity between enzyme and substrate,is nearly unaffected.The Km values at very alkaline pH are markedly elevated,indicating a conformational form which does not readily bind substrate.The pH profiles for kcat and kcat/Km are displaced towards the low pH side with increasing ionic strength.The ascending limb corresponds to the pK of the Asp-His charge relay system,decreasing with increasing ionic strength from 7.2 to 6.6 in the ES complex and from 6.8 to 6.6 in the free enzyme.Apparently substrate binding provokes a pK increase of the active His residue.The descending limb in the kcat profile could be described by a hypothetical pK varying from 11.5 to 10.7 but the activity decrease is probably due to enzyme inactivation.The alkaline limb of the kcat/Km profile is governed by a pK of 9.4 which is rather independent of the ionic strength and could be attributed to the B-chain terminal isoleucine ,forming a salt bridge with Asp 194 and stabilizing the active site conformation as proven for other serine proteases.Data analysis via a modified Debije function with appropriate estimates for the dielectric constant and the radius of the macro-ion can provide information on the charge density of the enzyme.
5
Asakura, S., N. Yoshida, and M. Matsuda. "MONOCLONAL ANTIBODIES AGAINST THROHBIN-ANTITHROMBIN III COMPLEX: EPITOPE SPECIFICITY AND EFFECT ON THROMBIN-ANTITHROMBIN III INTERACTION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643673.
Full textAbstract:
Among monoclonal antibodies (MCA´s) raised against human thrombin (T)-antithrombin m (AT) complex (TAT), two MCA´s designated as JITAT-16 and 17 with high affinity, Kd = 4.6nMand 4.1 nfi, respectively, were selected and characterized for specificity and functions. Their respective immunoglobulin subclasses are IgGi and IgG2a, and epitopes were found to be different from each Dther as shown by crisscross inhibition experiments. Immuno-alotting of normal plasma and serum electrophoresed on non-SDS aolyacrylamide gel showed that these antibodies reacted with normal serum but not with plasma. This was verified by an anzyme-linked differential antibody immunosorbent assay using aither one of the MCA´s as the first antibody and the other MCA labeled with peroxidase as the second one. By immunoblotting after SDS-PAGE, we found that both antibodies reacted with TAT, nut not with its respective nascent constituent, AT or T. However, they reacted with reactive site-cleaved AT (or thrombin-nodified AT, ATM) and also a complex of AT with activated factor K (Xa-AT). These results indicate that both of these antibodies recognize enzyme-treated forms of AT, including AT molecules :omplexed with enzymes reversibly or irreversibly as well as ATM. Jpon incubation of T with AT in the presence of JITAT-16, T activity remained nearly unchanged and formation of irreversible rAT did not proceed as expected. Moreover, AT was preferentially :onverted to ATM. When JITAT-16 was added after completion of FAT formation, however, neither recovery of T activity nor generation of ATM was observed. These findings were not obtained vhen JITAT-17 had been substituted for JITAT-16. These data suggest that JITAT-16 may have converted AT from an inhibitor to a substrate for T after having recognized a possible intermediate reversible complex of AT with T. Undoubtedly, in the presence of a polyclonal antibody against AT, neither TAT formation nor ATM neneration was observed at all. The mechanism of the unique Function of JITAT-16 has not been fully clarified as yet, but this antibody seems to give us new information on the kinetic study of TAT formation and ATM generation when AT was allowed to react with enzymes.
6
Visser, A., and D. G. Meuleman. "IRREVERSIBLE INHIBITION OF THE THROMBIN-MEDIATED SIGNAL TRANSFER." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644808.
Full textAbstract:
The inhibition of the thrombin-mediated signal transfer by a common irreversible inhibitor Z of the factor Xa complex (Xc a) and thrombin has been analysed for the two-step process of the Xc a-triggered formation of thrombin andthe consecutive splitting ok a thrombin-specific substrate S. Assuming that both proteolytic processes follow simple Michaelis—Menten kinetics, that the inhibition reactions are second-order and that the prothrombin and irreversible inhibitor are in excess it can be shown that:1. clotting time (tc) is inversely proportional to the time-averaged thrombin concentration2. the endpoint of the conversion of the thrombin specific substrate S reached at exhaustion of thrombin and the Xc a is inversely proportional to the square of the inhibitor concentration3. the continously monitored thrombin generation inhibition is a more sensitive assay than the classical two-stage thrombin generation inhibition assay4. the shift in the effective concentration range of the continuously monitored thrombin generation inhibition assay relative to the continuously monitored anti-Xa assay and to that of the continuously monitored anti-IIa assay, depends on the initial rate of formation of thrombin with the thrombin generation inhibition assay and the original enzyme concentrations of the anti-enzyme assays.It can further be shown that the above conclusions still hold when the Z-mediated (with Z = anti thrombin III e.g.) inhibitions are potentiated by heparin(oid)s.
7
Krishnaswamy, S., K. Jones, and K. Mann. "STOPPED-FLOW KINETICS OF PROTHROMBINASE COMPLEX ASSEMBLY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643279.
Full textAbstract:
Prothrombinase is the multi -component enzyme composed of factor Xa and the cofactor, factor Va, that assemble on a phospholipid surface in the presence of calcium ion. Fluoresence stopped-flow kinetic studies of prothrombinase assembly in the absence of the substrate, prothrombin were undertaken at saturating concentrations of calcium ion, using phospholipid vesicles (PCPS), factor Va and factor Xa modified at the active-site with the fluorophore dansyl-glutamylglycinylarginylchloro methylketone (DEGR-Xa). The substantial fluorescence enhancement of DEGR-Xa in the presence of factor Va, PCPS and calcium ion was used as a continuous measure of prothrombinase complex formation. The rate of complex formation studied under pseudo-first order conditions was most rapid when factor Va was reacted with the DEGR-Xa-PCPS binary complex. Under these conditions, the rate increased linearly with increasing PCPS concentrations and was independent of the concentration of factor Va. The reaction between Va and PCPS assessed by stopped-flow light scattering produced identical rates. The data indicate that the rate-limiting step in the reaction between Va and PCPS-bound DEGR-Xa is the initial formation of a Va-PCPS binary complex (8.4×107M−1s−1) followed by the very rapid reaction (>3×109M−1s−1) between PCPS-bound DEGR-Xa and Va. When complex formation was initiated by mixing DEGR-Xa and Va with PCPS or by mixing DEGR-Xa with Va-PCPS, the pseudo-first order rates were substantially lower. Under these conditions, increasing concentrations of factor Va inhibited the rate of prothrombinase complex formation which could be overcome by increasing PCPS concentrations. The data are consistent with the interpretation that the initial formation of the DEGR-Xa-PCPS complex is a prerequisite for prothrombinase assembly. The second order rate constant for the reaction between DEGR-Xa and PCPS (9.5×109M−1s−1 ) was independently determined by stopped-flow light scattering. Collectively, the data indicate that prothrombinase assembly proceeds via the initial formation of DEGR-Xa-PCPS and Va-PCPS binary complexes. The lipid-bound constituents then rapidly react via diffusion over the vesicle surface to form prothrombinase.(Supported by NIH grants HL-35058 and HL-34575)
8
Jordan, R. E., R. M. Nelson, and J. Kilpatrick. "KINETICS OF THE HEPARIN-DEPENDENT INACTIVATION OF ANTITHROMBIN BY NEUTROPHIL ELASTASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643898.
Full textAbstract:
The rate of inhibition of coagulation enzymes by antithrombin III is greatly increased by an interaction between the inhibitor and a limited, anticoagulantly-active subfraction of heparin molecules. We have observed that this same heparin sub-fraction also greatly stimulates the rate of inactivation of antithrombin by neutrophil elastase. Inactivation rates were increased several hundred-fold by catalytic amounts of the anticoagulantly active heparin species and were unaffected by the inactive heparin fraction or other glycosaminoglycans. Addition of catalytic amounts of elastase (1:400) to a solution of antithrombin in the presence of saturating levels of anticoagulantly-active heparin caused an approximately 30% decrease in the ultraviolet fluorescence emission of the inhibitor. The rate of fluorescence loss corresponded exactly with the loss of inhibitory activity in a parallel incubation under the same conditions. The use of the fluorescence technique for kinetic measurement of inactivation rates indicated a Km of less than 1 uM for the heparin-antithrombin complex substrate and a turnover of several hundred per minute per enzyme molecule. Although the specificity of the heparin effect appears to be at the level of its interaction with antithrombin, an elastase-heparin interaction could also be detected in kinetic analyses. Chromatographic studies employing immobilized heparin confirmed that elastase itself binds tightly to the complex carbohydrate. These results suggest a subtle mechanism, of potential relevance to thrombosis associated with inflammatory conditions, in which both heparin and elastase act catalytically to direct the inactivation of antithrombin. Since anticoagulantly-active heparin species are present on endothelial cells, the above mechanism could markedly affect the balance between procoagulant and anticoagulant processes on the usually non-thrombogenic endothelial surface.
9
MacGregor, I. R., N. A. Booth, N. R. Hunter, and B. Bennet. "QUANTIFICATION OF ENDOTHELIAL PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) ANTIGEN BY AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644450.
Full textAbstract:
An ELISA to measure PAI-1 antigen has been developed using PAI-1 purified from human endothelial cell conditioned medium and a monospecific antiserum raised against it in the rabbit. Test and standard samples diluted in assay buffer containing non-immune rabbit serum were incubated in microplate wells coated with anti-PAI-1 IgG. Then biotinylated anti-PAI-1 IgG was added to the wells followed by a streptavidin-biotinylated horseradish peroxidase complex. Tetramethylbenzidine was used as substrate and the optimised ELISA had a detection limit of 0.2 ng PAI-1 ml-1 sample, using purified PAI-1 of known concentration as a standard for the assay.PAI-1 antigen was readily detectable in human plasmas and higher concentrations were invariably detected in the corresponding whole blood sera.α2antiplasmin and antithrombin III which have been shown to have high degrees of sequence homology with PAI-J were undetectable in the ELISA at concentrations of 10 ug ml-1 . Sera from baboon and Rhesus and Cynomologus monkeys exhibited partial cross reactivity in the ELISA while no crossreactivities were observed with a wide range of non-primate sera that included dog, goat, cow, horse, pig and rat.A variety of human cell cultures were assayed for PAI-1 antigen. Endothelial cells from umbilical cord veins and arteries and from adult sapgenous veins secreted, typically,1-2 ug PAI-1 24-1 hr per 10 cells. This represented approximately 2-4% of the total secreted protein. Detectable but considerably lower levels of PAI-1 were secreted by keratinocytes and fibroblasts as well as a lung carcinoma cell line A549.No PAI-1 was detected in media conditioned by two melanoma cell lines, a breast carcinoma cell line MCF7 or a monocytic leukaemia JIII. The ELISA proved suitable for monitoring altered cellular PAI-1 metabolism, and in conjunction with functional assays, for investigating changes in PAI-1 specific activity.
10
Niederst, P. N., M. Asbach, M. Ott, and R. E. Zimmermann. "IN VITRO REACTION MODELS OF THROMBIN AND ITS PHYSIOLOGICAL INHIBITOR ANTITHROMBIN III IN THE PRESENCE OF HEPARIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644356.
Full textAbstract:
Antithrombin III (AT III) neutralizes thrombin and other serine proteases of plasma coagulation system by forming a stable 1:1 covalent complex. The inhibition rates are greatly increased by the potent catalyst heparin. The catalytic mechanism of heparin was studied in the presence of dextran sulfate (DS), a thrombin-binding sulfated Polysaccharid. DS did not influence the reaction of AT III with heparin and the amidolytic activity of thrombin, but preincubation with thrombin could inhibit the catalytic activity of heparin in the reaction of thrombin with AT III. We conclude that the reaction of heparin with enzyme and inhibitor, thus forming a ternary complex, is necessary for its catalytic activity.It is known that heparin also converts AT III from an inhibitor to a substrate for thrombin in a dose dependent manner. By cleavage of the reaction site bound Arg(385)-Ser(386) an AT III-fragment (MG 50000 d) occurs, which has a decreased affinity to heparin and does not inhibit F I la. At physiological ionic strength we have only measured a small percentage of AT 111-proteolysis (4%, 1 U/ml Hep). The extent of AT III-fragment formation could be enhanced by lowering the ionic strength (max 44%, 1 U/ml Hep., 1=0,02).