Plasticity of extended subsites facilitates divergent substrate recognition by Kex2 and furin

Abstract

Yeast Kex2 and human furin are subtilisin-related proprotein convertases that function in the late secretory pathway and exhibit similar though distinguishable patterns of substrate recognition. Although both enzymes prefer Arg at P1 and basic residues at P2, the two differ in recognition of P4 and P6 residues. To probe P4 and P6 recognition by Kex2p, furin-like substitutions were made in the putative S4 and S6 subsites of Kex2. T252D and Q283E mutations were introduced to increase the preference for Arg at P4 and P6, respectively. Glu255 was replaced with Ile to limit recognition of P4 Arg. The effects of putative S4 and S6 mutations were determined by examining the cleavage by purified mutant enzymes of a series of fluorogenic substrates with systematic changes in P4 and/or P6. Whereas wild type Kex2 exhibited little preference for Arg at P6, the T252D mutant and T252D/Q283E double mutant exhibited clear interactions with P6 Arg. Moreover, the T252D and T252D/Q283E substitutions altered the influence of the P6 residue on P4 recognition. We infer that cross-talk between S 4 and S6, not seen in furin, allows wild type and mutant forms of Kex2 to adapt their subsites for altered modes of recognition. This apparent plasticity may allow the subsites to rearrange their local environment to interact with different substrates in a productive manner. E255I-Kex2 exhibited significantly decreased recognition of P4 Arg in a tetrapeptide substrate with Lys at P1, although the general pattern of selectivity for aliphatic residues at P4 remained unchanged.