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Identification of a single amino acid as a primary determinant of prohormone convertase 1 cleavage specificity

Prohormone convertase 1 (PC1), present in a number of endocrine and neuronal cell types, is a serine endoproteinase that cleaves at specific dibasic sites to convert peptide precursors to their active form. Using a monomeric version of human proinsulin (DKP‐hPI) as a model, we discovered that an acidic cluster carboxy‐terminal to the cleavage site (cleavage sequence RR‐EAED, represented as P2P1‐P1′ P2′ P3′ P4′ with cleavage occurring between the P1 and P1′ amino acids) is required for efficient processing of the B chain/C peptide junction. Single amino acid deletions suggested that the acidic‐neutral sequence at P1′ P2′ is the most significant specificity determinant at this site. To continue our investigation, we replaced the P1′‐P4′ region with alanine residues (either individually or collectively) or with the P1′‐P4′ sequence from glicentin, a prohormone that is normally cleaved by PC2, but not by PC1. The alanine‐containing proinsulins were cleaved with efficiencies similar to the parent compound, while the glicentin‐substituted DKP‐hPI was cleaved quite poorly. To further define the specificity requirements, additional mutants were created. A reverse alanine mutation (A34E, introducing an acidic amino acid into the P2′ site) completely prevented PC1 cleavage at the B/C junction. In addition, sequential substitution of the glicentin sequence into the P1′‐P4′ region indicated that the P2′ amino acid (introduced as E33H/A34S‐DKP‐hPI) is responsible for diminishing the ability of PC1 to cut glicentin‐substituted proinsulin. We conclude that the presence of an aliphatic amino acid at the P2′ position is a major determinant of PC1 specificity.