Isolation, characterization, and preliminary X-ray diffraction data for a serine protease from Penicillium cyclopium.
The major extracellular protein of Penicillium cyclopium has been isolated from its culture media and purified by ammonium sulfate fractionation, gel, and ion-exchange chromatography. We show this secreted protein to be endopeptidase. The molecular weight is approximately 32,000, the pI is 5.0, and the pH optimum using a variety of protein and synthetic substrates is around 7.0. Inhibition studies show that the protease is not inhibited by pepstatin nor by p-chloromercuribenzoic acid, indicating, respectively, that it is not an aspartyl protease nor a thiol protease. Complete inhibition is observed, however, with phenylmethanesulfonyl fluoride. Three crystal forms suitable for high resolution x-ray diffraction studies have been obtained from this purified protease with reflections being observed to well beyond 3.0 A resolution. One form having a needle morphology is of the orthorhombic crystal class and has space group P2(1)2(1)2(1). The unit cell dimensions are a = 41.9 A, b = 43.2 A, and c = 111.5 A with 1 molecule of the protease occurring in the asymmetric unit. The second form grown at pH values less than 6.0 has a plate morphology, is of orthorhombic space group P2(1)2(1)2(1), and has unit cell dimensions a = 59.12 A, b = 62.33 A, and c = 70.62 A. The third form is polyhedral in habit, is also of space group P2(1)2(1)2(1), and appears when the pH of the mother liquor is greater than 7.0. The cell dimensions of this crystal form are a = 57.07 A, b = 58.82 A, c = 70.79 A, and again there is 1 molecule/asymmetric unit. Three-dimensional structural analysis by x-ray diffraction is now underway. All crystal forms are somewhat denser than the norm having mass to volume ratios of 1.58, 2.00, and 1.85 A3/dalton, respectively.
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D010450:Endopeptidases / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology*; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D014961:X-Ray Diffraction
D005656:Fungal Proteins; D011480:Protease Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases