The contribution of residues 192 and 193 to the specificity of snake venomserine proteinases

Snake venom serine proteinases, which belong to the subfamily of trypsin-li ke serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that o f the less specific enzyme trypsin, One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence iden tity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox veno m, specifically activates plasminogen to plasmin like tissue-type plasminog en activator (t-PA), even though it exhibits only 23% sequence identity wit h t-PA This study shows that TSV-PA, t-PA, and batroxobin are quite differe nt in their specificity toward small chromogenic substrates, TSV-PA being l ess selective than t-PA, and batroxobin not being efficient at all, The spe cificity of TSV-PA, with respect to t-PA and batroxobin, was investigated f urther by site directed mutagenesis in the 189-195 segment, which forms the basement of the S-1 pocket of TSV-PA and presents a His at position 192 an d a unique Phe at position 193, This study demonstrates that Phe(193) plays a more significant role than His(192) in determining substrate specificity and inhibition resistance. Interestingly, the TSV-PA variant F193G possess es a 8-9-fold increased activity for plasminogen and becomes sensitive to b ovine pancreatic trypsin inhibitor.