Antithrombin - Mechanism Of Action And Binding Of Heparin

1981 ◽  
Author(s):  
I Björk ◽  
U Lindahl
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inactivation ◽  
Substrate Complex ◽  
High Affinity ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Rate Limiting

Antithrombin inhibits a variety of serine proteases by forming equimolar, inactive complexes with the enzymes. The anti thrombin-thrombin complex, extensively studied as a model for complexes with other coagulation proteases, dissociates with a half-life of several days to free enzyme and a proteolytically modified inhibitor. It thus behaves like a kinetically stable enzyme-substrate complex. Several observations indicate that deacylation is the rate-limiting step. The active site of antithrombin, i.e. the bond slowly cleaved by the target enzyme, is the Arg-385/Ser-386 bond in the carboxy-terminal region of the protein. The formation of most anti thrombin-protease complexes is greatly accelerated by certain forms of heparin. These active molecules comprise about 1/3 of normal heparin preparations and bind with high affinity (K∼108 M-1) to the inhibitor, regardless of the size of the polysaccharide. The stoichiometry of binding is 1:1 for most heparin molecules, although some high-molecular-weight chains have two antithrombin binding sites. Evidence from spectroscopic and kinetic analyses suggests that the binding of high-affinity heparin induces a conformational change in antithrombin that probably is involved in the mechanism of the increased rate of enzyme inactivation. Oligosaccharides with high-affinity for anti thrombin have been isolated by affinity chromatography following partial deaminative cleavage of heparin with nitrous acid. The smallest such oligosaccharide obtained is an octasaccharide, in which a pentasaccharide sequence appears to comprize the actual antithrombin-binding site. This active sequence contains a unique, 3-O-sulfated glucosamine residue that does not appear to occur in other portions of the heparin molecule. In addition, two N-sulfate groups and probably at least one O-sulfate group within the pentasaccharide sequence are essential for high-affinity binding of heparin to antithrombin.

scholarly journals The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

1982 ◽  
Vol 203 (1) ◽  
pp. 149-153 ◽  
Author(s):  
P R Levison ◽  
G Tomalin
Keyword(s):  
Human Plasma ◽  
Methyl Esters ◽  
Plasma Kallikrein ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Kinetics Of Hydrolysis ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

scholarly journals Estimation of the dissociation constants of enzyme-substrate complexes from steady-state measurements. Interpretation of pH-independence of Km

1976 ◽  
Vol 153 (2) ◽  
pp. 455-461 ◽  
Author(s):  
A Cornish-Bowden
Keyword(s):  
Dissociation Constants ◽  
Michaelis Constant ◽  
Ph Profile ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Catalytic Constant ◽  
Almost All ◽  
Rate Limiting

If the Michaelis constant of an enzyme-catalysed reaction is independent of pH under conditions where the catalytic constant varies with pH, it is equal to the thermodynamic dissociation constant of the enzyme-substrate complex. This is true for realistic mechanisms in which binding and catalytic steps, are clearly distinguished, as well as for the simpler mechanisms that have been considered previously. It is also true for a mechanism in which a bell-shaped pH profile for the catalytic constant results from a change of rate-limiting step with pH. The relaxation time for ionization of a typical group in unbuffered solutions at 25 degrees C is of the order of 0.1 ms at the longest, and is much shorter in buffered solutions. Thus ionizations in almost all enzyme mechanisms can properly be treated as equilibria, provided that ionization is not accompanied by a slow, compulsory change in conformation.

Inhibition of Thrombin Generation in Plasma by Fibrin Formation (Antithrombin I)

2002 ◽  
Vol 88 (08) ◽  
pp. 253-258 ◽  
Author(s):  
N. B. de Bosch ◽  
A. Ruiz-Sáez ◽  
M. Echenagucia ◽  
A. Rodriguez-Lemoin ◽  
M. W. Mosesson
Keyword(s):  
New York ◽  
Binding Sites ◽  
Thrombin Generation ◽  
High Affinity ◽  
Fibrin Formation ◽  
Normal Plasma ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Inhibition Of Thrombin ◽  
Prothrombin Activation

SummaryThe adsorption of thrombin to fibrin during clotting defines “Antithrombin I” activity. We confirmed that thrombin generation in afibrinogenemic or in Reptilase defibrinated normal plasma was higher than in normal plasma. Repletion of these fibrinogen-deficient plasmas with fibrinogen 1 (‘γA/’γA), whose fibrin has two “low affinity” non-substrate thrombin binding sites, resulted in moderately reduced thrombin generation by 29-37%. Repletion with fibrinogen 2 (‘γ´/’γA), which in addition to low affinity thrombin-binding sites in fibrin, has a “high affinity” non-substrate thrombin binding site in the carboxy-terminal region of its ‘γ´ chain, was even more effective and reduced thrombin generation by 57-67%. Adding peptides that compete for thrombin binding to fibrin [S-Hir53-64 (hirugen) or ‘γ´ 414-427] caused a transient delay in the onset of otherwise robust thrombin generation, indicating that fibrin formation is necessary for full expression of Antithrombin I activity. Considered together, 1) the increased thrombin generation in afibrinogenemic or fibrinogen-depleted normal plasma that is mitigated by fibrinogen replacement; 2) evidence that prothrombin activation is increased in afibrinogenemia and normalized by fibrinogen replacement; 3) the severe thrombophilia that is associated with defective thrombin-binding in dysfibrinogenemias Naples I and New York I, and 4) the association of afibrinogenemia or hypofibrinogenemia with venous or arterial thromboembolism, indicate that Antithrombin I (fibrin) modulates thromboembolic potential by inhibiting thrombin generation in blood.Presented in part at the XVII Congress of the ISTH, Washington, D. C. (1)

scholarly journals Catalytic properties of alkaline phosphatase from pig kidney

1974 ◽  
Vol 141 (1) ◽  
pp. 283-291 ◽  
Author(s):  
Kunio Hiwada ◽  
Ernst D. Wachsmuth
Keyword(s):  
Alkaline Phosphatase ◽  
Enzymic Activity ◽  
Ph Optimum ◽  
Active Centre ◽  
Substrate Complex ◽  
Pig Kidney ◽  
Enzyme Substrate ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

The enzymic properties of alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes were studied. 1. It hydrolyses ortho- and pyro-phosphate esters, the rate limiting step (Vmax.) being independent of the substrate. It transphosphorylates to Tris at concentrations above 0.1m-Tris. 2. The pH optimum for hydrolysis was between 9.8 and 10. The pK of the enzyme–substrate complex is 8.7 for p-nitrophenyl phosphate and β-glycerophosphate. Excess of substrate inhibits the enzymic activity with decreasing pH. The pK of the substrate-inhibited enzyme–substrate complex, 8.7, is very similar to that for the enzyme–substrate complex. The pK values of the free enzyme appear to be 8.7 and 7.9. 3. Inactivation studies suggest that there is an essential tyrosine residue at the active centre of the enzyme. 4. The energy of activation (E) and the heat of activation (ΔH) at pH9.5 showed a transition at 24.8°C that was unaffected by Mg2+. 5. Kinetic and atomic-absorption analysis indicated the essential role of two Zn2+ ions/tetrameric enzyme for an ordered association of the monomers. Zn2+ in excess and other bivalent ions compete for a second site with Mg2+. Mg2+ enhances only the rate-limiting step of substrate hydrolysis. 6. Amino acid inhibition studies classified the pig kidney enzyme as an intermediate type of previously described alkaline phosphatases. It has more similarity with the enzyme from liver and bone than with that from placenta.

Photodegradation of hydrophobic disperse dyes and dye-bath effluents by silicadodecatungstate (SiW12O404-/5-) nanoparticles

2004 ◽  
Vol 49 (4) ◽  
pp. 171-176 ◽  
Author(s):  
I. Arslan-Alaton ◽  
S. Dogruel
Keyword(s):  
Chemical Oxidation ◽  
Reduced Form ◽  
Disperse Dyes ◽  
Power Ultrasound ◽  
Substrate Complex ◽  
Rate Limiting Step ◽  
Uv Photodegradation ◽  
The One ◽  
Rate Limiting ◽  
Decolorization Kinetics

Polyoxometalate (POM) silicadodecatungstic acid has been applied as a photochemical catalyst for the degradation of ®SETAPERS Black WNSP, a disperse dyestuff preparation widely used to dye polyester and polyamide fabrics. It could be demonstrated that the disperse dyestuff was photo-reduced by SiW12O405-, the one electron-reduced form of POM, as evidenced by Heteropolyblue (HPB) formation. For completion of the photochemical redox cycle, isopropanol (IsOH) was required. Acetone (Ac) served solely as an effective solute and photosensitizer; however this effect was suppressed in the presence of POM. Threshold (0.087 mM) and optimum (0.375 mM) POM concentrations existed and decolorization kinetics were inhibited upon the addition of dye auxiliary chemicals. Increasing the dyestuff concentration from 50 mg/L to 150 mg/L did not affect initial decolorization kinetics revealing that not the formation of the excited [POM-Substrate]* complex, but its reduction to HPB was the rate limiting step. POM-mediated, IsOH-assisted UV-photodegradation of disperse dyes and dye-baths is by far more effective than applying other, more well known chemical oxidation methods (O3, H2O2/UV, Power Ultrasound). Key to the action of POM redox catalysts is the feature that particularly heteropoly tungstates undergo facile re-oxidation to their original state, thus allowing regeneration of the photocatalyst, a feature that may become critical for real-scale application.

scholarly journals Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding

2019 ◽  
Vol 20 (24) ◽  
pp. 6203 ◽  
Author(s):  
Piero Leone ◽  
Michele Galluccio ◽  
Stefano Quarta ◽  
Ernesto Anoz-Carbonell ◽  
Milagros Medina ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Activity ◽  
Synthesis Rate ◽  
General Increase ◽  
Respiratory Chain Deficiency ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Acyl Coa Dehydrogenases ◽  
Rate Limiting ◽  
Fad Binding

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT “supermutant” of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.

A kinetic analysis of the processive enzyme Lactobacillus plantarum exoribonuclease

1977 ◽  
Vol 55 (7) ◽  
pp. 671-677 ◽  
Author(s):  
Kam-Fong Lam ◽  
David M. Logan
Keyword(s):  
Binding Energy ◽  
Lactobacillus Plantarum ◽  
Chain Length ◽  
Binding Sites ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Competitive Inhibitors ◽  
Kinetic Plots ◽  
Processive Enzyme ◽  
Almost All

Oligonucleotide chains consisting of adenosine residues and ranging from 1 to 70 residues in length have been tested as substrates or inhibitors with Lactobacillus plantarum exoribonuclease (EC 3.1.4.20). The kinetic constants V, Km, and Ki are all chain-length dependent. Ki decreases with increasing chain length to a minimum for oligonucleotides seven residues in length and then begins to increase slightly. Kinetic plots indicate that the oligonucleotides are almost all competitive inhibitors of poly A hydrolysis. However, the oligonucleotide (Ap)3A > p probably leads to mixed inhibition. The enzyme is unable to retain its processivity when it hydrolyzes short oligonucleotides such as (Ap)2A and (Ap)3A. It is proposed that L. plantarum exoribonuclease possesses seven binding sites for the polynucleotide. When the enzyme is bound to a long-chain-length substrate the complex is stabilized by a binding energy of about 8 Kcal/mol. After cleavage of the terminal nucleotide the remaining binding energy is still sufficient to maintain an enzyme–substrate complex. The shortened nucleotide chain is moved relative to the enzyme to re-form the seven-bond association by a gradient of energy of about 1.7 Kcal/mol for the change from six to seven bonds.

DEVELOPMENT OF HOMOLOGOUS IMMUNOLOGICAL ASSAYS FOR HUMAN PARATHYROID HORMONE

1980 ◽  
Vol 85 (1) ◽  
pp. 161-170 ◽  
Author(s):  
R. M. MANNING ◽  
G. N. HENDY ◽  
S. E. PAPAPOULOS ◽  
J. L. H. O'RIORDAN
Keyword(s):  
Parathyroid Hormone ◽  
Trichloroacetic Acid ◽  
Terminal Region ◽  
Human Parathyroid Hormone ◽  
Antibody Assay ◽  
Amino Terminal ◽  
High Affinity ◽  
Carboxy Terminal Region ◽  
Immunological Assays ◽  
Carboxy Terminal

SUMMARY Antisera to a trichloroacetic-acid precipitate of human parathyroid hormone (PTH) were produced in goats. Two of these antisera (G36 and G31) were of high affinity, and the bovine and porcine hormones were less reactive. Synthetic peptides containing the amino-terminal region of human PTH reacted with both antisera; the 1–34 peptide (PTH-(1–34)), with the sequence proposed by Niall, Sauer, Jacobs, Keutmann, Segre, O'Riordan, Aurbach & Potts in 1974, was more reactive than that having the sequence proposed by Brewer, Fairwell, Ronan, Sizemore & Arnaud in 1972. The antisera were further characterized with a number of other native and synthetic fragments of human PTH and reacted poorly with fragments from the carboxy-terminal region of the molecule. Since the amino-terminal fragments did not account for all the immunoreactivity, it is assumed that the antisera had some recognition sites for the central part of the molecule. Highly purified human PTH-(1–84) was labelled with 125I and radioimmunoassays were developed using this tracer and antiserum G36. To avoid the problems associated with labelling human PTH with 125I, a labelled antibody assay was developed with G36 and an immunoadsorbent consisting of human PTH-(1–34) (sequence of Niall et al.) coupled to cellulose. A sensitive homologous amino-terminal specific assay was developed in this way.

scholarly journals The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

2019 ◽  
Author(s):  
Jennifer Nill ◽  
Tina Jeoh
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Cellulose Hydrolysis ◽  
Reaction Rates ◽  
Hydrolysis Kinetics ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Substrate Properties ◽  
Hydrolysis Of

AbstractInterfacial enzyme reactions require formation of an enzyme-substrate complex at the surface of a heterogeneous substrate, but often multiple modes of enzyme binding and types of binding sites complicate analysis of their kinetics. Excess of heterogeneous substrate is often used as a justification to model the substrate as unchanging; but using the study of the enzymatic hydrolysis of insoluble cellulose as an example, we argue that reaction rates are dependent on evolving substrate interfacial properties. We hypothesize that the relative abundance of binding sites on cellulose where hydrolysis can occur (productive binding sites) and binding sites where hydrolysis cannot be initiated or is inhibited (non-productive binding sites) contribute to rate limitations. We show that the initial total number of productive binding sites (the productive binding capacity) determines the magnitude of the initial burst phase of cellulose hydrolysis, while productive binding site depletion explains overall hydrolysis kinetics. Furthermore, we show that irreversibly bound surface enzymes contribute to the depletion of productive binding sites. Our model shows that increasing the ratio of productive- to non-productive binding sites promotes hydrolysis, while maintaining an elevated productive binding capacity throughout conversion is key to preventing hydrolysis slowdown.

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