Substrate Specificity of the Widely Expressed Subtilisin-like Proprotein Convertases

Abstract

Limited endoproteolysis plays a key role in the activation and maturation of proteins that traverse the secretory pathway. Subtilisin-like Proprotein Convertases (SPCs) are a family of eukaryotic endoprotease that carry out limited endoproteolysis in the secretory pathway. SPCs activate a diverse array of proproteins like clotting factors, enzymes, hormones, and also proteins involved in infectious and non-infectious diseases. SPCs activate proteins by recognizing a cleavage sequence generally described as N-Arg-X-X-Arg-C (with each amino acid designated as N-P4-Pe-P2-P1-C). However, each enzyme has its unique substrate preferences, which likely impart particular physiological and pathological roles. We examined the substrate preferences of widely expressed SPCs (furin, PACE4, PC5, and PC7) at the P2 position of substrates, using both qualitative and quantitative analysis. We found that all the enzymes show a similar pattern of substrate preferences, but at the same time they also have unique characteristics. Furin at pH 6.0 showed a broader range of substrate preferences than at pH 7.0. PACE4 showed stringent substrate specificity at P2 position. PC7 was less efficient over the entire range of substrates. To understand which amino acids might make PACE4 different from furin, we examined the S2 subsite (amino acids in the enzyme interacting with teh P2 position in substrates of PACE4 by creating three mutants called E94D, D90T, DM. The results of D90T suggest that aspartic acid at position 90 of PACE4 helps mediate P2 sequence requirements. Qualitative results did not necessarily agree with teh quantitative results likely because of overall change in the structural conformation of the substrate proteins.