Studies on prolyl endopeptidase from shakashimeji (Lyophyllum cinerascens): purification and enzymatic properties.
High prolyl endopeptidase (post-proline cleaving enzyme) [EC 3.4.21.26] activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes. Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase. The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4. The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators. It was estimated that at least five subsites were concerned with the enzyme-substrate binding. Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes. The enzyme hydrolyzed TRH at the carboxyl side of the proline residue. The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Yoshimoto
T
T
School of Pharmaceutical Sciences, Nagasaki University.
Sattar
A K
AK
Hirose
W
W
Tsuru
D
D
D001487:Basidiomycota / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D010450:Endopeptidases / Q000302:isolation & purification*; D007531:Isoflurophate / Q000037:antagonists & inhibitors; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases; D013237:Stereoisomerism
D007531:Isoflurophate; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases