PRIMARY STRUCTURE OF ASPERGILLOPEPSIN-I DEDUCED FROM NUCLEOTIDE-SEQUENCE OF THE GENE AND ASPARTIC ACID-76 IS AN ESSENTIAL ACTIVE-SITE OF THE ENZYME FOR TRYPSINOGEN ACTIVATION

The coding region of the aspergillopepsin I (EC 3.4.23.18) gene occupi es 1340 base pairs of the genomic DNA and is separated into four exons by three introns. The predicted amino-acid sequence of aspergillopeps in I consists of 325 residues and is 32% and 27% homologous with those of human pepsin and calf chymosin. The cDNA of the gene prepared from mRNA has been cloned and expressed in yeast cells. To identify the re sidue of the substrate binding pocket in determining the specificity o f aspergillopepsin I towards basic substrates, this residue was replac ed with a serine residue by site-directed mutagenesis. The mutation is a single amino-acid change, Asp-76 converted to Ser-D76S, in the enzy me. The striking feature of this is that only the trypsinogen activati ng activity was destroyed. We therefore concluded that Asp-76 is the b inding site towards basic substrates.