Studies on the molecular properties and the structure-function relationships of carboxyl proteases of novel type

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1. The complete amino acid sequence of a carbowxl protease of novel type (Proctase A) produced by the fungus, Aspergillus niger var. macrosporus, has been determined by conventional methods of protein chemistry. Further, the gene and cDNA of Proctase A have been cloned and the base sequence of the coding region of the cDNA has been elucidated.2. The substrate specificity toward various protein and peptide substrates of Proctase A has been elucidated and the active-site residues have been explored by chemical modification. In addition, the cDNA for proproctase A could be expressed in E. coli and shown to be autocatalytically activated under acidic conditions.3. Proctase A could be crystallized and a preliminary X-ray-study on it has been performed. On the other hand, two-dimensional NMR and circular dichroism studies have revealed that Proctase A has a high beta-sheet structure and a rigid core structure in the molecule.4. Proctase A has been shown to be denatured rapidly and irreversibly at pH 6-7 with simultaneous dissociation of the two peptide chains.5. A unique pepstatin-insensitive acid protease (M 54K) has been purified from the plasmodia of a true slime mold, Physarum polycephalum, and shown to have a unique two chain structure and substrate specificity.6. Procathepsin E has been purified from the gastric mucosa of human stomach, and its NH_2-terminal amino acid sequence and the site of carbohydrate attachment have been clarified. Further, procathepsin E has been shown to be autocatalytically activated and to have significant proteolytic activity at neutral pH with rather narrower substrate specificity.7. The gene for Drosophila copia protease could be expressed in E. coli, giving an active enzyme. Its cleavage specificity toward the precursor protein has been elucidated, and the importance for activity of an aspartic acid residue at the putative active site has been demonstrated by site-directed mutagenesis.