CHARACTERIZATION OF AN EXOCELLULAR SERINE-THIOL PROTEINASE ACTIVITY IN PARACOCCIDIOIDES-BRASILIENSIS

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 P-1 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 (P-3) and arginine (P-2) in this substrate, with a decrease in the K-m 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.