The contribution of residues 192 and 193 to the specificity of snake venom serine proteinases.
Snake venom serine proteinases, which belong to the subfamily of trypsin-like serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that of the less specific enzyme trypsin. One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence identity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom, specifically activates plasminogen to plasmin like tissue-type plasminogen activator (t-PA), even though it exhibits only 23% sequence identity with t-PA. This study shows that TSV-PA, t-PA, and batroxobin are quite different in their specificity toward small chromogenic substrates, TSV-PA being less selective than t-PA, and batroxobin not being efficient at all. The specificity of TSV-PA, with respect to t-PA and batroxobin, was investigated further 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 and 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 possesses a 8-9-fold increased activity for plasminogen and becomes sensitive to bovine pancreatic trypsin inhibitor.
Braud
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S
Unité des Venins, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Parry
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MA
Maroun
R
R
Bon
C
C
Wisner
A
A
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000378:metabolism; D012103:Batroxobin / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D003435:Crotalid Venoms; D006023:Glycoproteins; D011994:Recombinant Proteins; D010649:Phenylalanine; D006639:Histidine; D005340:Fibrinogen; D010958:Plasminogen; D007611:Aprotinin; D012103:Batroxobin; D010960:Plasminogen Activators; D012697:Serine Endopeptidases; C093329:TSV-PA protein, Trimeresurus stejnegeri; D002918:Chymotrypsin; D010959:Tissue Plasminogen Activator