Purification and characterization of an extracellular prolyl endopeptidase from Agaricus bisporus.
Prolyl endopeptidase [EC 3.4.21.26] was purified to homogeneity from the culture filtrate of Agaricus bisporus by a procedure that comprised ammonium sulfate fractionation, anion-exchange chromatographies on DEAE-Toyopearl and DEAE-Sephadex, hydroxylapatite chromatography, and high-performance liquid chromatography (HPLC) on a TSKgel G 2000 SW column. The overall activity recovery was 8.6%. The enzyme was most active at or around pH 7.5 and was stable in the range of pH 5-9 when checked with Z-Gly-Pro-beta-naphthylamide as a substrate. The isoelectric point of the enzyme was about 4.8. The enzyme was a monomeric protein of molecular weight 78,000 +/- 2,000 as judged by gel permeation chromatography on Sephadex G-150 and electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gel. The enzyme hydrolyzed Pro-X bonds and at least five subsites (S3, S2, S1, S1', and S2') were found to be involved in enzyme-substrate binding. Among them, S2, S1, and S1' subsites of the enzyme showed high stereospecificity. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), Z-Gly-Pro-CH2Cl, Z-Pro-prolinal, Z-Pro-pyrrolidine, Z-Thiopro-pyrrolidine, Z-Pro-thiazolidine, Z-Thioprothiazolidine, and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenyl-methylsulfonyl fluoride (PMSF), E-64, iodoacetamide, or metal chelators. Although the A. bisporus enzyme showed no immunological cross reaction with anti-bovine prolyl endopeptidase antiserum, the other characteristics were quite similar to those of mammalian and plant enzymes.
Sattar
A K
AK
School of Pharmaceutical Sciences, Nagasaki University.
Yamamoto
N
N
Yoshimoto
T
T
Tsuru
D
D
D000363:Agaricales / Q000201:enzymology*; D000364:Agaricus / Q000201:enzymology*; D000595:Amino Acid Sequence; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology; D007460:Iodoacetamide / Q000494:pharmacology; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D010618:Phenanthrolines; D004492:Edetic Acid; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D007460:Iodoacetamide