Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida.
Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis-Menten constant (K(m)) for Chromozym ELA (approximately 245 microM) is much higher than those for the thrombin (approximately 90 microM) and trypsin (approximately 60 microM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1'-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55-60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys(77)-Arg(78), Arg(342)-Met(343), Ala(444)-Ala(445) and Arg(557)-Ile(558). The site Arg(557)-Ile(558) is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala(444)-Ala(445) released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the Aalpha chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val(21)-Glu(22) site.
Zhao
Jing
J
Lab of Visual Information Processing, Institute of Biophysics, Center for Brain and Cognitive Sciences, Baiao Pharmaceuticals Beijing C.L., Beijing, China.
Li
Li
L
Wu
Cen
C
He
Rong-Qiao
RQ
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology*; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D005340:Fibrinogen; D010958:Plasminogen; D010450:Endopeptidases; C083421:lumbrokinase