A Novel Venombin B fromAgkistrodon contortrix contortrix:  Evidence for Recognition Properties in the Surface around the Primary Specificity Pocket Different from Thrombin

Abstract

A novel thrombin-like enzyme (named contortrixobin) has been purified to homogeneity from the venom of Agkistrodon contortrix contortrix by affinity chromatography on arginine-Sepharose, anionic exchange chromatography, and HPLC. The complete amino acid sequence has been determined by Edman degradation and by mass spectral analysis of peptides generated by enzymatic cleavage. A microheterogeneity at the level of residue 234 has been detected, as demonstrated by peptides differing for the occurrence of Pro234 ( approximately 85%) or Asp234 ( approximately 15%). Contortrixobin (i) has six disulfide bonds whose sequence positions have been determined by mass spectrometry and (ii) does not contain carbohydrates in its structure. As expected, the 234 residue sequence of contortrixobin exhibits strong homology with snake venom serine proteases acting on either fibrinogen or other blood coagulation components. The interaction of contortrixobin with chromogenic substrates indicates a higher specificity for arginine over lysine in the primary subsite and a faster attack to ester than amides. The hydrolytic activity of contortrixobin is strongly inhibited by diisopropyl fluorophosphate and to a less extent by phenylmethylsulfonyl fluoride, benzamidine, and 4', 6-diamidino-2-phenylindole; hirudin (a specific alpha-thrombin inhibitor) as well as basic pancreatic trypsin inhibitor has a small effect on contortrixobin's catalytic properties. Contortrixobin (i) preferentially releases fibrinopeptide B from human fibrinogen, (ii) activates blood coagulation Factors V and XIII with a rate 250-500-fold lower than human alpha-thrombin, and (iii) does not induce thrombocyte aggregation, intracytoplasmatic calcium ion increase in platelets, and activation of Factor VIII. Evidence for biorecognition properties different from thrombin is also reported.

Snake venom thrombin-like enzymes: from reptilase to now

“…Since then, several SVTLEs were described in the literature not only in the same species, such as A. caliginosus [11], A. c. contortrix [12], A. halys blomhoffii [13], A. h. pallas [14,15], B. atrox [16,17], B. jararaca [18], B. jararacussu [19], T. stejnegeri [20] and T. flavoviridis [21 -23], but also in different species, e. g. A. halys [24], A. h. brevicaudus stejneger [25], A. h. ussuriensis [26,27], A. 844 H. C. Castro et al Snake venom thrombin-like enzymes saxatilis [28], B. alternatus [29], B. lanceolatus [30], T. mucrosquamatus [31], T. elegans [32,33], T. jerdonii [34,35], T. stejnegeri [36] and different genus Gloydius (G. ussuriensis and G. shedaoensis) [37,38].…”

Molecular Basis for the Partition of the Essential Functions of Thrombin among Snake Venom Serine Proteinases

“…It follows then that the molecular mechanism of recognition and binding of ligands by SVTLEs is different from those of thrombin and not mediated by allosteric transitions. Some of the factors contributing then in a combined fashion to these differences include: (1) the absence of the cleft formed by the 60-loop of thrombin (making the active site more accessible); (2) the number of acidic residues lining the primary-specificity pocket; (3) the number and type of residues in the apolar binding site near the catalytic center; (4) the number and location of the basic residues constituting the ESI [33].…”

A Brief Appraisal on Russell’s Viper Venom (Daboia russelii russelii) Proteinases

“…Russelobin was found to be significantly inhibited by thrombin inhibitors such as heparin and weakly inhibited by antithrombin-III unlike other thrombin-like snake venom proteinases such as batroxobin, cerastocytin, cerastotin, and contortrixobin (Mukherjee and Mackessy 2013). The difference in the active site of Russelobin compared to other SVSPs was more prominent due to the fact that Russelobin was not inhibited by tosyl phenylalanyl chloromethyl ketone (TPCK) and tosyl lysine chloromethyl ketone (TLCK) (Mukherjee and Mackessy 2013), whereas other SVSPs were inhibited by TPCK as well as TLCK (Marrakchi et al 1997;Amiconi et al 2000). Russelobin demonstrated a high degree of selectivity toward fibrinogen without detectable proteolysis of other plasma proteins, suggesting that fibrinogen is the primary physiological substrate for Russelobin.…”