Initial sites of insulin cleavage and stereospecificity of carboxyl proteinases from Aspergillus sojae and Pycnoporus coccineus.
Initial cleavage sites of native insulin at a pH of about 3 and stereospecificity were investigated by fungal carboxyl proteinases (EC 3.4.23.6) from ASpergillus sojae, a species of fungi imperfecti, and Pycnoporus coccineus (formerly designated Trametes sanguinea), a wood deteriorating Basidiomycete, respectively. Fungal carboxyl proteinases were used as a model of vertebrate insulin degradation. A. sojae carboxyl proteinase I primarily hydrolyzed two peptide bonds located on the surface of native insulin monomer, the B16-B17 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds, and secondarily the buried bonds, A15-A16 (Gln-Leu), B15-B16 (Leu-Tyr) and B14-B15 (ala-Leu), at pH 3.2 and 30 degree C. The initial cleavage sites of A. sojae carboxyl proteinases I towards native insulin were not identical with the initial cleavage sites towards the oxidized B chain of insulin. P. coccineus carboxyl proteinase Ia selectively hydrolyzed B14-B15 (Ala-Leu), B16-B17 (Tyr-Leu) and B24-B25 (Phe-Phe) bonds in the native insulin at pH 2.7. Based on these findings we suggest that the stereospecificity of the fungal carboxyl proteinases is similar to that of cathepsin D (EC 3.4.23.5), and that the synthesis and degradation of insulin may occur in microorganisms.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001230:Aspergillus / Q000201:enzymology*; D001487:Basidiomycota / Q000201:enzymology*; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin; D010446:Peptide Fragments / Q000032:analysis; D013379:Substrate Specificity
D000596:Amino Acids; D007328:Insulin; D010446:Peptide Fragments; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases