Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates

1986/07/01 by K Ohta, Kaoru Ohta, K K Makinen +3 · 227 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · Dentistry · Health Professions · #Biochemistry #Casein #Chemistry #Chromatography #Diisopropyl fluorophosphate #Enzyme #Gel electrophoresis #Oral and gingival health research #Oral microbiology and periodontitis research #Phenylmethylsulfonyl Fluoride #Polyacrylamide gel electrophoresis #Protease #Protein Hydrolysis and Bioactive Peptides #Sephadex #Sodium dodecyl sulfate #Trypsin

paper · pdf · doi:10.1128/iai.53.1.213-220.1986

published in Infection and Immunity 53(1), 213-220 (American Society for Microbiology)

openalex publication_date 1986/07/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/08/01

Abstract

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.

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