title: Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis.
abstract: An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.
authors.affiliation: Departmento de Biofísica, Escola Paulista de Medicina, São Paulo, Brazil.
authors.forename: A K
authors.initials: AK
authors.lastname: Carmona
authors.forename: R
authors.initials: R
authors.lastname: Puccia
authors.forename: M C
authors.initials: MC
authors.lastname: Oliveira
authors.forename: E G
authors.initials: EG
authors.lastname: Rodrigues
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: L R
authors.initials: LR
authors.lastname: Travassos
mesh_terms: D000595:Amino Acid Sequence; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010228:Paracoccidioides / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D010455:Peptides; D011480:Protease Inhibitors; D012697:Serine Endopeptidases