Specificity of acid proteinase A from Aspergillus niger var. macrosporus towards B-chain of performic acid oxidized bovine insulin.
1. A comparative study on the mode of action of two highly purified acid endopeptidases (EC 3.4.-) from Aspergillus niger var. macrosporus, acid proteinase A and B, on the B-chain of performic acid oxidized insulin was performed, putting emphasis on the quantitative analysis of the effects of enzyme A. Acid proteinase A behaved very specifically towards the substrate and hydrolyzed four peptide bonds exclusively: three major sites, where hydrolysis proceeded rapidly and almost completely, Asn3-Gln4, Glu13-Ala14, and Tyr26-Thr27; and a minor one, Gly20-Glu21, at which hydrolysis was much slower. 2. The effects of four protease inhibitors, pepstatin, diazoacetyl-D,L-norleucine methyl ester/Cu(II), di-isopropyl phosphorofluoridate, and 1,2-epoxy-3-(p-nitrophenozy) propane on acid proteinases A and B were studied. Acid proteinase A preparations, treated with the former two inhibitors, were used to establish that the major sites of attack were really affected by enzyme A and not by contaminating proteinase B.
D000596:Amino Acids / Q000032:analysis; D001230:Aspergillus / Q000201:enzymology*; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D005561:Formates; D007328:Insulin; D010446:Peptide Fragments / Q000378:metabolism*; D011480:Protease Inhibitors
D000596:Amino Acids; D005561:Formates; D007328:Insulin; D010446:Peptide Fragments; D011480:Protease Inhibitors