scholarly journals Secondary-site binding of Glu-plasmin, Lys-plasmin and miniplasmin to fibrin

1981 ◽  
Vol 197 (3) ◽  
pp. 619-628 ◽  
Author(s):  
Elisabeth Suenson ◽  
Sixtus Thorsen
Keyword(s):  
Tranexamic Acid ◽  
Binding Site ◽  
Active Site ◽  
Marked Change ◽  
Secondary Site ◽  
Minor Importance ◽  
Kunitz Inhibitor ◽  
Pancreatic Trypsin Inhibitor ◽  
Human Plasminogen ◽  
Plasmin Inhibitor

Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1–790)]. This binding is decreased by α2-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, α2-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu1–Lys76, Glu1–Arg67or Glu1–Lys77)display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, α2-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys.170, 300–305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of α2-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu1–Val441) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by α2-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than α2-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.

scholarly journals Two conformationally vicinal thiols at the active site of Leishmania donovani adenosine kinase

1996 ◽  
Vol 316 (2) ◽  
pp. 439-445 ◽  
Author(s):  
Tapan K. BAGUI ◽  
Mallika GHOSH ◽  
Alok K. DATTA
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Leishmania Donovani ◽  
Marked Change ◽  
Adenosine Kinase ◽  
Inactivation Kinetics ◽  
Cysteine Residues ◽  
Schematic Model ◽  
Hydrophobic Domain ◽  
Modified Enzyme

Inactivation of adenosine kinase (Adk) from Leishmania donovani correlates with the modification of two conformationally vicinal cysteine residues. In contrast, Adk from hamster liver, despite being sensitive to monothiol-blocking reagents, was insensitive to dithiol modifiers. Inactivation kinetics and substrate-protection studies along with double-modification experiments successively with N-ethylmaleimide in the presence of Ado and sodium m-arsenite–2,3-dimercaptopropanol or diazenedicarboxylic acid bis-N,N´-dimethylamide supported assignment of the two thiols at the Ado-binding site. Cystine bridge formation impaired the ability of the modified enzyme to bind to the substrate. Tryptophan fluorescence of the enzyme was quenched after modification by dithiol-blocking reagents with concomitant loss of activity. However, treatment of the enzyme with methylmethanethiosulphonate (MMTS) led to complete inactivation without a marked change in protein fluorescence. Ado protected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern–Volmer quenching analysis of the native and Ado-complexed enzyme suggested that, of the four tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and dithiol-modified enzyme in the presence of either acrylamide or KI indicated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the results suggest important function(s) for the cysteine residue(s). A schematic model is proposed to explain the inactivation of the enzyme by both monothiol- and dithiol-blocking reagents.

scholarly journals Kinetics of plasmin inhibition in the presence of a synthetic tripeptide substrate. The reaction with pancreatic trypsin inhibitor and two forms of α 2-plasmin inhibitor

1981 ◽  
Vol 199 (1) ◽  
pp. 121-127 ◽  
Author(s):  
L C Petersen ◽  
I Clemmensen
Keyword(s):  
Trypsin Inhibitor ◽  
Order Reaction ◽  
Secondary Site ◽  
Kinetic Properties ◽  
Pseudo First Order Reaction ◽  
First Order ◽  
Pancreatic Trypsin Inhibitor ◽  
Binding Capability ◽  
Kinetics Of ◽  
Plasmin Inhibitor

The progressive inhibition of plasmin by pancreatic trypsin inhibitor and by alpha 2-plasmin inhibitor in the presence of D-valyl-L-leucyl-L-lysine 4-nitroanilide was investigated. The kinetics with plasmin were compared with those with miniplasmin. The kinetic properties of two functionally different forms of alpha 2-plasmin inhibitor described by Clemmensen [(1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen. D., Wiman, B & Verstraete, M., eds.), pp 131-136, Elsevier, Amsterdam] were characterized. The two forms differ in their plasminogen-binding capability, and this difference can account for a difference in secondary site interaction suggested from the kinetics. The binding of inhibitor to miniplasmin is a simple pseudo-first-order reaction with both pancreatic trypsin inhibitor and the two alpha 2-plasmin inhibitor forms. Such simple kinetics are also observed for the reaction between plasmin and the non-plasminogen-binding form of alpha 2-plasmin inhibitor. More complicated kinetics are obtained for the reaction between plasmin and the alpha 2-plasmin inhibitor form that binds to plasminogen. With both forms of the alpha 2-plasmin inhibitor, a complex stable to acetic acid/urea and gel electrophoresis is present and fully developed 15 s after initiation of the reaction with plasmin.

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

1998 ◽  
Vol 17 (7) ◽  
pp. 699-712 ◽  
Author(s):  
Dennis E. Epps ◽  
Roger A. Poorman ◽  
Gary L. Petzold ◽  
Christopher W. Stuchly ◽  
Alice L. Laborde ◽  
...  
Keyword(s):  
Binding Site ◽  
Fluorescent Probes ◽  
Active Site

scholarly journals Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

2004 ◽  
Vol 382 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Pakorn WINAYANUWATTIKUN ◽  
Albert J. KETTERMAN
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Anopheles Dirus ◽  
Steady State Kinetics ◽  
Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

scholarly journals Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

2017 ◽  
Vol 73 (12) ◽  
pp. 651-656 ◽  
Author(s):  
Taichi Mizobuchi ◽  
Risako Nonaka ◽  
Motoki Yoshimura ◽  
Katsumasa Abe ◽  
Shouji Takahashi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Active Site ◽  
Metal Binding ◽  
Bivalve Mollusc ◽  
Active Site Residues ◽  
X Ray ◽  
Aspartate Racemase ◽  
Scapharca Broughtonii

Aspartate racemase (AspR) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that is responsible for D-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-binding site, but a different orientation of the metal-binding site. Superposition of the structures of SbAspR and of rat SR bound to the inhibitor malonate reveals that Arg140 recognizes the β-carboxyl group of the substrate aspartate in SbAspR. It is hypothesized that the aromatic proline interaction between the domains, which favours the closed form of SbAspR, influences the arrangement of Arg140 at the active site.

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