Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conversion of factor X to factor Xa results in release of a heavily glycosylated activation peptide. Analysis of protease-digested glycopeptides derived from the activation peptides of bovine and human blood coagulation factor X allowed the identification of sites of the O-linked oligosaccharide chains in these peptides. Glycopeptides were prepared from the activation peptides by digestion with chymotrypsin or Staphylococcus aureus V8 protease. By combined analysis of amino acid sequence and sialic acid content, we found that bovine factor X had an O-linked oligosaccharide chain linked to Thr26, and human factor X had four carbohydrate-attachment sites, namely, O-glycosidic linkages to Thr17 and Thr29, respectively, and N-glycosidic linkages to Asn39 and Asn49, respectively, in their activation peptides. The O-linked carbohydrate-attachment sites were identified since the yields of phenylthiohydantoin derivatives of amino acids that corresponded to their residues were increased during amino acid sequencing after deglycosylation of the glycopeptides with sialidase and O-glycanase. The effect of deglycosylation of bovine factor X1 was investigated with factor-X-activating enzyme from Russell's viper venom or extrinsic Xase (factor VIIa/tissue factor/phospholipid) by examining the activation rates of derivatives of factor X prepared using O-glycanase, sialidase, and/or N-glycanase. The removal of O-linked carbohydrate resulted in a decrease in the rate of activation. It appears that carbohydrate residues in factor X play an important role in the activation of the zymogen.
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PMID:Identification of O-linked oligosaccharide chains in the activation peptides of blood coagulation factor X. The role of the carbohydrate moieties in the activation of factor X. 824 61

Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF-bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365-374{216-225}) of the so-called activation domain and the 170-loop (residues 313-322{170A-175}) succeeding the TF-binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active-site inhibitor. Based on MD simulations, a key switch of the TF-induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S(1) and S(3) substrate pockets, the activation pocket, and N-terminal insertion.
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PMID:A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation. 1738 32