Hydrolysis of peptide bonds of the oxidized B-chain of insulin by Endothia parasitica protease

Abstract

The relative rates of hydrolysis of the peptide bonds of the oxidized B-chain of insulin by Endothia parasitica protease have been determined at pH 3.60 and at a substrate concentration of 1.07 × 10 −3 m and an enzyme concentration of 1.95 × 10 −6 m. The Phe 24Phe 25, bond is hydrolyzed at a maximum rate followed by hydrolysis of the Tyr 16Leu 17 and Gln 4His 5 bonds. The Leu 11Val 12 and Asn 3Gln 4 bonds are hydrolyzed at slower rates. The LeunVal 12 bond appears to be considerably more resistant to hydrolysis in the peptide 5–16 than in the intact oxidized B-chain. The Leu 15-Tyr 16 bond is very slowly hydrolyzed in peptide 5–16 but there is no evidence for hydrolysis of this bond in the intact oxidized B-chain. Phe 25 is slowly hydrolyzed from the peptide 25–30 and the bond involving Gly 20Glu 21 is slowly hydrolyzed in peptide 12–24 and/or peptide 17–24. Thus the enzyme has specificity for the hydrophobic regions of the oxidized B-chain. The specificity of Endothia parasitica protease on the oxidized B-chain of insulin is similar to that of pepsin in that bonds involving Gln 4His 5, Leu 11Val 12, Leu 15Tyr 16, Tyr 16Leu 17, Phe 24Phe 25 and Phe 25Tyr 26 are hydrolyzed by both enzymes. Endothia parasitica protease hydrolyzes bonds involving Asn 3Gln 4 and Gly 20Glu 20, not attacked by pepsin, but fails to hydrolyze bonds involving Phe 1Val 2, Glu 13Ala 14, Ala 14Leu 15 and Gly 23Phe 24, hydrolyzed by pepsin. Thus, Endothia parasitica protease is more specific in its action on the oxidized B-chain of insulin than is pepsin.