Crotalase, a fibrinogen-clotting snake venom enzyme: Primary structure andevidence for a fibrinogen recognition exosite different from thrombin

Crotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradat ion. The resulting amino add sequence, VIGGDEC NINEHRFLVALYDYWSQLFLCGGTLINN EWVLTAAHCDRTHI LIYVGVHDRSVQFDKEQRRFPKEKYFFDCSNNFTKWDKDIM LIRLNKPVSYSEHIAPLS LPSSPPIVGSVCRAMGWGQTTSPQET LPDVPHCANINLLDYEVCRTAHPQFRLPATSRTLCAGVLEG GIDTCN RDSGGPLICNGQFQGIVFWGPDPCAQPDKPGLYTK VFDHLDWIQSIIAGEKTVNCP, is characteristi c of a serine proteinase. Comparison with thrombin, the physiological fibri nogen-clotting enzyme, showed that thrombin's fibrinogen-recognition exosit e (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibi tor, had no effect on crotalase. Spatial modeling of crotalase yielded a po ssible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85 , Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also la cks thrombin's YPPW loop, as well as its functionally important ETW 146-148 , and its heparin-binding site. The enzyme contains a single asparagine-lin ked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme's total molecular weight of 29,027. The calculated a bsorbance of crotalase at 280 nm, 1%, cm(-1) is 15.2.