Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates.
An enzyme from Treponema denticola that hydrolyzes a synthetic trypsin substrate, N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), was purified to near homogeneity, as judged by gel electrophoresis. The molecular weight of the enzyme was estimated to be ca. 69,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ca. 50,000 by gel filtration on Sephadex G-100. The pH optimum for the hydrolysis of BAPNA was around 8.5. The enzyme was heat labile and irreversibly inactivated at low pH values. Enzyme activity was enhanced by Ca2+, Mg2+, and Ba2+ but inhibited by Mn2+, Hg2+, Co2+, and Zn2+. Metal chelators and sulfhydryl reagents had no effect on this activity. The enzyme was inhibited by certain protease inhibitors such as diisopropyl fluorophosphate, N-alpha-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethylchloromethyl ketone, alpha-1-antitrypsin, and soybean trypsin inhibitor. The Km values for BAPNA and N-alpha-benzoyl-L-arginine ethyl ester were 0.05 and 0.12 mM, respectively, and the Vmax values were higher than those observed with trypsin. Although the purified enzyme hydrolyzed some low-molecular-weight synthetic trypsin substrates, it did not hydrolyze casein, hemoglobin, azocasein, azocoll, bovine serum albumin, or gelatin. Thus, this enzyme is probably not a protease but is capable of hydrolyzing ester, amide, and peptide bonds involving the carboxyl group of arginine and lysine.
D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D002413:Cations, Divalent; D003773:Dental Plaque / Q000382:microbiology; D004950:Esterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D014208:Treponema / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D002413:Cations, Divalent; D014361:Trypsin Inhibitors; D001586:Benzoylarginine Nitroanilide; D004950:Esterases; D014357:Trypsin