The proprotein convertase PC1/3 is synthesized as a large precursor that undergoes proteolytic processing of the signal peptide, the propeptide and ultimately the COOH-terminal tail, to generate the mature form. The propeptide is essential for protease folding and although cleaved by an autocatalytic process, it remains associated with the mature form acting as an auto-inhibitor of PC1/3. To further assess the role of certain residues in its interaction with its cognate enzyme, we performed an alanine scan on two PC1/3 propeptide potential cleavable sites (RRSRR54 and KR62) and an acidic region DDD67 conserved among species. Upon incubation with PC1/3, the ensuing peptides exhibit equal inhibitory potency, lower potency or higher potency than the WT propeptide. The Ki values calculated varied between 0.15 and 16.5 nM. All but one mutant exhibited a tight binding behavior. In order to examine the specificity of mutants, we studied their reactivity towards furin, a closely related convertase. The mutation of certain residues also affects the inhibition behavior toward furin yielding propeptides exhibiting Ki ranging from 0.2 to 24 nM. Mutant propeptides exhibited against each enzyme either different mode of inhibition, enhanced selectivity in the order of 40 fold for one enzyme or high potency with no discrimination. Hence, we demonstrate through single amino acid substitution that it is feasible to modify the inhibitory behavior of propeptides towards convertases in such a way as to increase or decrease their potency, modify their inhibitory mechanisms as well as increase their selectivity.