SummaryAs in the previous inventory (93), the term “thrombin-like”, continues to mean direct “fibrinogen-clotting”. The present tabulation adds 32 publications to the earlier one and focuses mainly on biochemical properties. Broader treatment of these enzymes, such as medical and other biological applications, can be found in a number of review articles and chapters (9, 69, 90, 110, 118).

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During the amino acid sequence determination of crotalase (EC 3.4.21.30), the. thrombin-like enzyme from the venom of Crotals mantes-(eastern diamondback rattlesnake), we found that, in addition to the expected structural homologywith bovine thrombin (EC 3.4.21.5), there was even greater homology with porcine pancreatic kallikrein (EC 3.4.21.8). In exploring further the similarities between crotalase and kallikrein, several striking observations were made. First, crotalase was rapidly and specifically inhibited by the tripeptide affinity labeling derivative prolylphenylalanylarginine chloromethyl ketone, which is known to be a specific inhibitor of kallikrein. Second, NaDodSO4 acrylamide gel electrophoresis revealed that crotalase cleaves the plasma kallikrein-susceptible bonds. in human high molecular weight kininogen, producing an intermediate with procoagulant activity. Crotalase-catalyzed cleavage of high molecular weight kininogen also liberates kinin as evidenced by rat blood pressure bioassay. Finally,. crotalase exhibits substrate specificity not only for the thrombin chromogenic substrate S-2238 but also for the kallikrein substrates S-2302 and S-2266. Interestingly, one of the other reactions catalyzed by, plasma kallikrein, the activation of plasminogen, was not one of the activities exhibited-by crotalase.Crotalase (EC 3.4.21.30), the thrombin-like enzyme from the venom of Crotalus adamanteus (eastern diamondback rattlesnake) has been purified to homogeneity (1-4). Physicochemical characterization of crotalase indicated that it contains a single polypeptide chain with a molecular weight of 33,000 (1). Although analysis for carbohydrate indicated that the enzyme is a sialoglycoprotein, removal of the sialic acid does not appear to alter its enzymatic activity (2). Crotalase clots fibrinogen and plasma anticoagulated with EDTA or heparin (5). In contrast to thrombin (EC 3.4.21.5), crotalase cleaves only fibrinopeptide A from fibrinogen (6) and it does not activate factor XIII (4). There are also other subtle differences in specificity between these two enzymes (4, 7).The enzymatic properties of crotalase have been investigated, and it has been found that the enzyme exhibits esterase activity on small, basic amino acid esters (1). Both coagulant and esterase activities are inhibited-rapidly and simultaneously by diisopropyl fluorophosphate. These findings are consistent with crotalase being a member of the serine protease family (1,3,4). Interestingly, the chloromethyl ketone ofN-aAtosyl-L-lysine also inhibits both esterase and coagulant activities but only partially and with a very slow reaction rate.During recent investigations aimed at elucidating the primary structure of crotalase (8,9), it became evident that there was significant homology between the sequence ofcrotalase (9) and the known primary structure of thrombin (10). Of more interest, however, was the even greater degree of homology between crotalase (9) and glandular kallikrein (EC 3.4.21.8) (11). Although these findings were unex...

SummaryCrotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence, VIGGDEC NINEHRFLVALYDYWSQLFLCGGTLINNEWVLTAAHCDRTHI LIYVGVHDRSVQFDKEQRRFPKEKYFFDCSNNFTKWDKDIM LIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRAMGWGQTTSPQET LPDVPHCANINLLDYEVCRTAHPQFRLPATSRTLCAGVLEG GIDTCNRDSGGPLICNGQFQGIVFWGPDPCAQPDKPGLYTK VFDHLDWIQSIIAGEKTVNCP, is characteristic of a serine protein-ase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin’s fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin’s YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked 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 absorbance of crotalase at 280 nm, 1%, cm-1is 15.2.

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