scholarly journals Evidence for common structural changes in thrombin induced by active-site or exosite binding

1993 ◽  
Vol 290 (3) ◽  
pp. 665-670 ◽  
Author(s):  
M A A Parry ◽  
S R Stone ◽  
J Hofsteenge ◽  
M P Jackman
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Limited Proteolysis ◽  
Intrinsic Fluorescence ◽  
Thrombin Inhibitors ◽  
Site Specific ◽  
Fluorescence Measurements ◽  
Fold Reduction ◽  
Flexible Surface ◽  
Fractional Increase

The gamma-loop of thrombin is a flexible, surface-accessible loop in free thrombin that appears to be one of several sites participating in the interaction of the enzyme with macromolecular substrates and inhibitors. Using limited proteolysis and intrinsic fluorescence measurements, we have studied changes in thrombin structure induced by small, site-specific ligands. Binding of a C-terminal peptide of hirudin to the anion-binding exosite of thrombin induced a structural change in the gamma-loop, which caused a 6-fold reduction in the susceptibility of the enzyme to limited proteolysis by elastase and chymotrypsin. Binding of several active site-specific thrombin inhibitors conferred an even greater protection from proteolysis at the gamma-loop. For example, the covalent complex of thrombin with D-Phe-Pro-Arg-CH2Cl was 95-fold less susceptible to cleavage by chymotrypsin than the free enzyme. Furthermore, binding of either exosite or active-site probes induced a common intrinsic fluorescence change in thrombin (a fractional increase of 0.13). These results are surprising because crystallographic studies indicate that direct contact between the bound probes and relevant residues of the gamma-loop is very unlikely. Thus we have identified an allosteric interaction that couples the active site of thrombin to the gamma-loop. An interaction of this nature may be one way in which thrombomodulin modulates the reactivity of thrombin.

The Ability of Thrombin Inhibitors to Reduce the Thrombin Activity Generated in Plasma on Extrinsic and Intrinsic Activation

1997 ◽  
Vol 77 (03) ◽  
pp. 498-503 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Anion Binding ◽  
Thrombin Inhibitors ◽  
Coagulation System ◽  
Thrombin Activity ◽  
Continuous Registration ◽  
High Concentrations ◽  
20 Nm ◽  
Generation Test

SummaryIn a thrombin generation test with continuous registration of thrombin activity in plasma we studied the ability of a variety of thrombin inhibitors of different type and mechanism of action to influence the activity of thrombin after activation of the coagulation system. Depending on the inhibitor, the peak of thrombin activity is delayed and/or reduced.By blocking the active site of generated thrombin inhibitors cause a concentration dependent reduction of the thrombin peak and inhibit feed-back reactions of thrombin resulting in a delay of thrombin generation. Highly potent synthetic active-site directed inhibitors (Ki ≤ 20 nM) reduce the thrombin activity formed in plasma after extrinsic or intrinsic activation with the same efficiency (IC50 0.1 - 0.6 μM) as hirudin. The delay and reduction of thrombin generation by inhibitors of the anion-binding exosite 1 of thrombin is only attributed to an inhibition of feed-back reactions of thrombin. For a 50% reduction of thrombin activity in plasma by this type of inhibitors relatively high concentrations were determined.

scholarly journals The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 648
Author(s):  
del Prado ◽  
Santos ◽  
Lázaro ◽  
Salas ◽  
de Vega
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Structural Changes ◽  
Binding Capacity ◽  
Dna Polymerases ◽  
Crystallographic Structure ◽  
Genome Replication ◽  
Terminal Protein ◽  
Phi29 Dna Polymerase

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

scholarly journals Cheminformatics Modeling of Closantel Analogues for Treating River Blindness

2020 ◽  
Author(s):  
Melaine A. Kuenemann ◽  
Phyo Phyo Zin ◽  
Sravya Kuchibhotla ◽  
Denis Fourches
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Parasitic Nematode ◽  
Chemical Space ◽  
Therapeutic Agents ◽  
Binding Modes ◽  
River Blindness ◽  
Structure Activity ◽  
Potential Binding ◽  
Biological Target

<p></p><p>Onchocerciasis (also known as river blindness<i>)</i> is a neglected tropical disease caused by the <i>Onchocerca volvulus</i> parasitic nematode. Currently, the only approved drug for treating this disease is ivermectin, which is a broad-spectrum antiparasitic agent. However, signs of resistance towards ivermectin have started to emerge. New therapeutic agents are thus urgently needed. The OvCHT1 chitinase enzyme from <i>O. volvulus</i> has been established as a relevant biological target for combatting river blindness. The veterinary anthelmintic drug closantel has been found to be a potent, micro-molar OvCHT1 inhibitor. Herein, we investigated the chemical space of closantel and all its synthesized analogues, focusing on the analysis of their potential binding modes towards OvCHT1. First, we conducted an unsupervised hierarchical clustering to group highly similar analogues and explore structure-activity relationships. Second, we conducted a structure-based molecular docking to predict and study the binding modes of all 57 closantel analogues in the active site of OvCHT1. Third, we screened more than 4 million lead-like compounds from the ZINC library to identify other structurally similar ligands that could potentially bind to OvCHT1. The cheminformatics analysis of the closantel analogues illustrated how minor structural changes in closantel analogues can impact their OvCHT1 activity.</p><p></p>

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

1992 ◽  
Vol 12 (9) ◽  
pp. 3757-3765
Author(s):  
J W Chen ◽  
B R Evans ◽  
S H Yang ◽  
H Araki ◽  
Y Oshima ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Substrate Binding ◽  
Point Mutations ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Site Specific ◽  
Arginine Residues ◽  
Active Site Tyrosine ◽  
Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

Active site-directed thrombin inhibitors: α-Hydroxy-acyl-prolyl-arginals: New orally active stable analogues of D-Phe-Pro-Arg-H

Peptides 1994 ◽  
1995 ◽  
pp. 343-344
Author(s):  
S. Bajusz ◽  
E. Barabás ◽  
I. Fauszt ◽  
A. Fehér ◽  
Gy. Horváth ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Inhibitors ◽  
Orally Active
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