MOLECULAR MODELING OF THE SUBSTRATE-SPECIFICITY OF PROHORMONE CONVERTASES SPC2 AND SPC3

In this paper we describe the results of molecular modeling of the str uctures of the active sites of two subtilisin-like prohormone converta ses (SPCs), SPC2 (PC2) and SPC3 (PC1/PC3). These enzymes are members o f a recently discovered family of cellular proteases involved in the p rocessing of precursor proteins. Although these proteases all possess catalytic domains similar to the bacterial subtilisins no tertiary str uctural data from x-ray analysis are yet available. We have shown that despite the high structural homology of the subtilisins and the SPCs, the structure of the loop which lies immediately below the active sit es differs due to the presence of a cis-peptide bond (Tyr(167)-pro(168 )) in this loop in the subtilisins and its absence in the SPCs. Accord ingly, we have proposed a new alignment for the amino acid sequences o f the SPCs in this region. Both SPC2 and SPC3 participate in the proce ssing of prohormones at dibasic cleavage sites, typically Lys-Arg or A rg-Arg. To investigate the structural basis of the substrate specifici ty of these SPCs, we have carried out molecular mechanic calculations of the optimal arrangement and interactions of peptide substrates cont aining several residues of arginine or lysine, i.e. Arg, Ala-Ala Ala-A rg, Arg-AZa-Ala-Arg, Arg-Ala-Arg-Arg, Arg-Ala-Lys-Arg, in the putative active sites. Such subtilisin-based modeling has allowed us to identi fy those negatively charged residues, Asp and Glu, in the S1, S2, and S4 subsites, which can directly interact with basic residues in the su bstrates via formation of salt bridges and thereby contribute to the s ubstrate selectivity of the SPCs.