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, hydrox-ylapatite 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-/9-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, Si, S, ', and S/) were found to be involved in enzyme-substrate binding. Among them, S2, S1( and S, ' subsites of the enzyme showed high stereospecificity. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), Z-Gly-Pro-CH2C1, Z-Pro-prolinal, Z-Pro-pyrrolidine, Z-Thiopro-pyrrolidine, Z-Pro-thiazolidine, Z-Thiopro-thiazolidine, 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. © 1990 COPYRIGHT, 1990 BY THE JOURNAL OF BIOCHEMISTRY.