Partial purification and some properties of pyroglutamyl peptidase from Enterococcus faecalis.
Pyroglutamyl peptidase was partially purified from Enterococcus faecalis ATCC 19433 by anion-exchange chromatography, gel filtration and salting out after lysis of cell walls with N-acetylmuramidase. Pyroglutamyl peptidase was purified 46-fold with a yield of about 2% based on the total activity of the crude extract. The molecular mass of the bacterial enzyme was estimated to be about 82 kD by gel filtration. The pl of the enzyme was 4.2 and the optimum pH and temperatures for the reaction were 7.2-7.5 and 35-45 degrees C, respectively. The enzyme was relatively stable below 45 degrees C, but almost all the activity was lost after heat-treatment at 55 degrees C for 15 min. The apparent K(m) value for pyroglutamyl-beta-naphthylamide was 0.55 mM. The bacterial enzyme specifically cleaved pyroglutamyl residues from the amino termini of pyroglutamyl compounds, such as Pyr-Asn-Gly, Pyr-His-Gly, Pyr-Ala-Glu, Pyr-Ala, neurotensin, thyrotropin-releasing hormone and bradykinin-potentiator B. However, human IgG and Bence Jones protein, which are high-molecular-mass proteins, were not hydrolysed. Neither derivatives of free amino acids, such as Ala-, Gly-, Pro- and Leu-p-nitroanilide, nor benzoyl-DL-Arg-p-nitroanilide were hydrolysed. The activity was strongly inhibited by thiol-blocking reagents (p-CMB, N-ethylmaleimide, monoiodoacetic acid). In addition, protease inhibitors, such as TLCK and PMSF, reduced the activity by 54 to 73%. These results suggest that the bacterial enzyme is a cysteine protease with sulphydryl residues in its active site and, possibly, histidine or serine residues near the active site.
Mineyama
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Department of Oral Microbiology, School of Dentistry at Niigata, Nippon Dental University, Japan.
Saito
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K
D000814:Aniline Compounds / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D019216:Metals, Heavy / Q000737:chemistry; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000737:chemistry; D011751:Pyroglutamyl-Peptidase I / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000737:chemistry
D000814:Aniline Compounds; D019216:Metals, Heavy; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; C019498:4-nitroaniline; D006026:Glycoside Hydrolases; C026255:endo-N-acetylmuramidase; D011751:Pyroglutamyl-Peptidase I