Initial sites of insulin cleavage and stereospecificity of carboxyl proteinases from Aspergillus sojae and Pycnoporus coccineus

Abstract

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°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 I a 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.