HETEROLOGOUS EXPRESSION AND SITE-DIRECTED MUTAGENESIS STUDIES ON THE ACTIVATION MECHANISM AND THE ROLES OF THE BASIC RESIDUES IN THE PROSEGMENT OF ASPERGILLOPEPSINOGEN-I

To study the structure/function relationship of the prosegment of aspa rtic proteinase, a putative proform of aspergillopepsin I (or proteina se B) from Aspergillus niger var. macrosporus was expressed by Escheri chia coli, refolded in vitro, and purified. The conversion of the puri fied proenzyme (aspergillopepsinogen I, proproteinase B) into the acti ve mature form occurred at pH less than or equal to 4.5 and was comple tely inhibited by pepstatin A, a specific inhibitor for aspartic prote inase, suggesting autoprocessing. The N-terminus of this mature form w as Glu67 (numbering in preproform), which was different from the N-ter minal Ser70 of native proteinase B although there was no significant d ifference in enzymatic activity. During the conversion, two intermedia tes were observed on SDS/PAGE, indicating a stepwise mechanism. The Ly s56-Phe57 sequence seems to be a counterpart of the Lys-Tyr pair highl y conserved in the prosequences of aspartic proteinases. When the muta nt proenzyme (K56N), in which Lys56 was replaced with Asn by site-dire cted mutagenesis, was allowed to refold under various conditions, no s ignificant potential activity could be obtained. Proproteinase B was a lso expressed by Bacillus brevis HPD31. This system required no in vit ro refolding to obtain potentially active proenzyme, which was secrete d into the culture medium (30-120 mg/l) and had the same properties wi th that obtained by the E. coil system. The K56N mutant prepared by th is system also had no potential activity, and was rapidly digested by incubation with native proteinase B, suggesting that the mutant did no t fold correctly. On the other hand, the K56R mutant (Lys56-->Arg) was potentially active. These results indicated that Lys56 is essential f or the folding through electrostatic interaction with the catalytic As p residues in the active site although it may be replaced with Arg. In the presence of a low concentration of pepstatin A, an incompletely p rocessed form with N-terminal Ser53 was obtained. Further, the R52Q (A rg52-->Gln) mutant showed no processing but was converted to the activ e mature form by incubation with the native enzyme. Therefore, the cle avage between Arg52 and Ser53 is considered to be the initial and esse ntial step of the autoactivation. The R26Q, K27Q, R36Q, K40Q, R42Q, an d K66Q mutants were also potentially active. The K66Q mutant was proce ssed to a form with N-terminal Ala55.