Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum.
Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency but didn't show any reactivity for the gamma form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30 degrees C and the maximum fibrinolytic activity was at 55 degrees C.
Kim
J H
JH
Department of Chemistry, Sangii University, Wonju, Korea.
Kim
Y S
YS
D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D005337:Fibrin; D005340:Fibrinogen; D008666:Metalloendopeptidases