Contribution of the 60 Loop To Substrate and Inhibitor Specificity of Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1734-1734 ◽  
Author(s):  
Alireza R. Rezaie
Keyword(s):  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Inhibitor Specificity ◽  
Active Site Pocket ◽  
Catalytic Function ◽  
Target Molecules

Relative to chymotrypsin, the 60-loop of thrombin contains 8–9 insertion residues which are believed to be partly responsible for the restricted substrate and inhibitor specificity of thrombin. Previous deletion of 3–4 residues of this loop (des-PPW and des-YPPW) dramatically impaired the activity of thrombin toward antithrombin, protein C and fibrinogen, implicating a key role for the productive interaction of these residues with the target macromolecules. To further investigate the role of this loop, we expressed a mutant of thrombin in which all 8 insertion residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, we discovered that des-60-loop thrombin cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired ~2–4-fold. Further studies revealed that the reactivity of des-60-loop with antithrombin is not impaired, but rather improved ~2-fold. Remarkably, the mutant could also activate prothrombin to thrombin. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket; however, its productive interaction with the target molecules may not be as critical for the catalytic function of thrombin as has been speculated in previous reports.

Deletion of the 60-loop provides new insights into the substrate and inhibitor specificity of thrombin

2005 ◽  
Vol 93 (06) ◽  
pp. 1047-1054 ◽  
Author(s):  
Likui Yang ◽  
Alireza Rezaie
Keyword(s):  
Mammalian Cells ◽  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Structural Data ◽  
Inhibitor Specificity ◽  
Direct Binding ◽  
Target Molecules ◽  
Inhibition Studies

SummaryStructural data have indicated that the 60-loop of thrombin with 8–9 insertion residues is responsible for the restricted substrate and inhibitor specificity of thrombin. However, previous deletion of 3–4 residues of this loop (des-PPW and des-YPPW) did not widen the specificity of thrombin, but further restricted it. The partial deletion of this loop also dramatically impaired the reactivity of thrombin with antithrombin (AT), protein C and fibrinogen, implicating a role for the productive interaction of the 60-loop with the target macromolecules. To further investigate the role of this loop, a mutant of thrombin was expressed in mammalian cells in which all 8 residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, it was discovered that the des-60-loop mutant cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired ~2–4-fold. Direct binding and AT inhibition studies in the presence of heparin revealed that the affinity of heparin for interaction with exosite-2 of des-60-loop thrombin was impaired, though the reactivity of the mutant with AT and other plasma serpins was not impaired, but rather improved ~2-fold. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket, but its productive interaction with the target molecules may not be as critical as has been speculated in previous reports.

scholarly journals Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

2006 ◽  
Vol 396 (2) ◽  
pp. 355-362 ◽  
Author(s):  
Fatbardha Varfaj ◽  
Julie Neuberg ◽  
P. Vincent Jenkins ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
High Concentration ◽  
Subunit Dissociation ◽  
Factor Viiia ◽  
Km Value ◽  
Type Factor

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate ∼25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated ∼9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated ∼2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a Km value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than Ki values (∼9–18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341–345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.

Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity

2019 ◽  
Vol 167 (3) ◽  
pp. 315-322
Author(s):  
An-Ning Feng ◽  
Chih-Wei Huang ◽  
Chi-Huei Lin ◽  
Yung-Lung Chang ◽  
Meng-Yuan Ni ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
N Terminus ◽  
Key Enzyme ◽  
The Stability

Abstract 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

scholarly journals Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type β-lactamase

RSC Advances ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 5325-5337 ◽  
Author(s):  
Lubna Maryam ◽  
Shamsi Khalid ◽  
Abid Ali ◽  
Asad U. Khan
Keyword(s):  
Amino Acid ◽  
Significant Role ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Beta Lactamase ◽  
Amino Acid Residues ◽  
Catalytic Function ◽  
Close Proximity

Mutations of amino acid residues present near active site decrease the catalytic efficiency of beta lactamase enzymes.

Protective role of activated protein C in lung and airway remodeling

2004 ◽  
Vol 32 (Supplement) ◽  
pp. S262-S265 ◽  
Author(s):  
Koji Suzuki ◽  
Esteban Cesar Gabazza ◽  
Tatsuya Hayashi ◽  
Haruhiko Kamada ◽  
Yukihiko Adachi ◽  
...  
Keyword(s):  
Protein C ◽  
Airway Remodeling ◽  
Activated Protein C ◽  
Protective Role

scholarly journals Inhibition of staurosporine-induced apoptosis of endothelial cells by activated protein C requires protease-activated receptor-1 and endothelial cell protein C receptor

2003 ◽  
Vol 373 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Laurent O. MOSNIER ◽  
John H. GRIFFIN
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Cell Protein ◽  
Protease Activated Receptor 1 ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Dose Dependent

In a model of staurosporine-induced apoptosis using EAhy926 endothelial cells, inhibition of apoptosis by activated protein C was dose-dependent and required the enzyme's active site, implicating activated protein C-mediated proteolysis. Consistent with this implication, both protease-activated receptor-1 (PAR-1) and endothelial cell protein C receptor (EPCR) were required for the anti-apoptotic effects of activated protein C.

Sepsis and the Role of Activated Protein C

2004 ◽  
Vol 24 (6) ◽  
pp. 40-45
Author(s):  
Janice Tazbir
Keyword(s):  
Protein C ◽  
Activated Protein C

scholarly journals Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 642-647 ◽  
Author(s):  
K Murakami ◽  
K Okajima ◽  
M Uchiba ◽  
M Johno ◽  
T Nakagaki ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Vascular Injury ◽  
Active Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Myeloperoxidase Activity ◽  
Granulocyte Elastase ◽  
Pulmonary Vascular Permeability ◽  
Elastase Inhibitor

We investigated the effect of activated protein C (APC) on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats to investigate the possible usefulness of APC as a treatment for adult respiratory distress syndrome. Intravenously administered LPS (5 mg/kg) significantly increased pulmonary vascular permeability. APC prevented the LPS-induced increase in pulmonary vascular permeability observed at 6 hours. Heparin plus antithrombin III (ATIII) and active site-blocked factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, inhibited LPS-induced coagulopathy but did not prevent LPS-induced pulmonary vascular injury. LPS-induced pulmonary vascular injury was significantly attenuated in rats with nitrogen mustard-induced leukocytopenia and in rats treated with ONO-5046, a potent granulocyte elastase inhibitor. Administration of LPS also increased pulmonary accumulation of leukocytes, as evaluated by measurement of myeloperoxidase activity in the lungs. APC significantly reduced LPS- induced increases in pulmonary accumulation of leukocytes at 1 hour. Neither ATIII plus heparin nor DEGR-Xa inhibited leukocyte accumulation. Active site-blocked APC (DIP-APC) prevented neither the LPS-induced pulmonary accumulation of leukocytes nor the LPS-induced increase in pulmonary vascular permeability. These results suggest that the mechanism of APC inhibition of LPS-induced pulmonary vascular injury was independent of its anticoagulant activity and was related to its ability to inhibit accumulation of leukocytes. In addition, these findings suggest that the serine protease activity of APC may be essential to its inhibitory effect on LPS-induced pulmonary accumulation of leukocytes and subsequent pulmonary vascular injury.

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