VaSP1, catalytically active serine proteinase from Vipera ammodytes ammodytes venom with unconventional active site triad

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• 作者：Tihana Kurtovi膰 ; Marija Brgles ; Adrijana Leonardi ; Maja Lang Balija ; Tamara Sajevic ; Igor Kri啪aj ; G眉nter Allmaier ; Martina Marchetti-Deschmann ; Beata Halassy
• 关键词：Snake venom serine proteinase ; Non-canonical catalytic triad ; Vipera ammodytes ; Mass spectrometry
• 刊名：Toxicon
• 出版年：January, 2014
• 年：2014
• 卷：77
• 期：Complete
• 页码：93-104
• 全文大小：1380 K

文摘

VaSP1, a serine proteinase from Vipera ammodytes ammodytes venom, is a glycosylated monomer of 31.5聽kDa, as determined by MALDI mass spectrometry, showing multiple isoelectric points between pH 6.5 and pH 8.5. Partial amino acid sequencing of VaSP1 by Edman degradation and MS/MS analysis identified sequences which allowed its classification among the so-called snake venom serine proteinase homologues, members of the peptidase S1 family, however being devoid of the canonical catalytic triad. Only few representatives of this group have been identified so far with just two of them characterised in detail at the protein level. Despite substitution of His57 with Arg, VaSP1 possesses proteolytic activity which can be inhibited by Pefabloc, benzamidine, Zn²⁺ ions, DTT and trypsin inhibitor II, a Kunitz/BPTI group member. It hydrolyses N_伪-benzoyl-Phe-Val-Arg-p-NA, exhibiting Michaelis-Menten behaviour with K_m聽=聽48.2聽渭M and V_m聽=聽0.019聽nM聽s^鈭?. The pH for optimal activity on tested substrate is around 9.0. VaSP1 also cleaves insulin B-chain, digesting it at positions His¹⁰-Leu¹¹_, Ala¹⁴-Leu¹⁵ and Tyr¹⁶-Leu¹⁷. Furthermore, the novel serine proteinase is active towards wide array of proteins involved in haemostasis where its degradation of fibrinogen, fibrin, prothrombin, factor X and plasminogen in聽vivo probably results in depletion of coagulation factors in blood circulation. The possibility that VaSP1 possesses anticoagulant properties has been further indicated by its ability to prolong prothrombin time and activated partial thromboplastin time.