Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase. To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions. The circular dichroism spectra of the mutant pro-and mature enzymes were essentially the same as those of the wildtype pro-and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.The regular aspartic proteinase family (1-5) includes such enzymes as pepsin, chymosin, cathepsins D and E, renin, and fungal and retroviral pepsins. The family is homologous both in primary and tertiary structures so they are thought to share a common ancestor in evolution. Generally, members of the aspartic proteinase family are composed of two homologous domains, each containing a catalytic aspartyl residue in a consensus sequence of Asp-(Thr/Ser)-Gly, in close proximity to enable catalytic function (6). The proteinases are often characterized by susceptibility to specific inhibitors such as pepstatin A (7), 1,2-epoxy-3-(p-nitrophenoxy)propane (8), and the diazoacetyl-DL-norleucine methyl ester in the presence of cupric ions (9).In addition to the aspartic proteinases, there exist some distinct families of aspartic or acid proteinases that are not homologous, having neither apparent internal sequence homology nor the consensus Asp-(Thr/Ser)-Gly sequence present in the pepsin-type aspartic proteinases. Aspergillopepsin II is unique in the primary structure of known proteins with the exception of scytalidopepsin B (20) and the EapB and EapC proteins from C. parasitica (14). Aspergillopepsin II consists of two polypeptide chains, a 39-residue light chain and a 173-residue heavy chain (11), which are derived from a precursor polypeptide of 282 residues (21). Although this two-chain structure is different from the one-chain structures of scytalidopepsin B and the EapB and EapC proteins, a 44 -65% identity in amino acid sequence indicates that these proteins have been derived in evolution from the same ancestral protein and presumably share common catalytic residues and mechanisms. Recently Barrett et al. (22) have classified...
