Specificity characterization of the α-mating factor hormone by Kex2 protease.

Abstract Kex2 is a Ca 2+ -dependent serine protease from S. cerevisiae . Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S ′ of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR↓EADQ-EDDnp and Abz-YKR↓EADQ-EDDnp based on the pro-α-mating factor sequence. The specificity of Kex2 due to basic residues at P 1 ′ is affected by the type of residue in the P 3 position. Some residues in P 1 ′ with large or bulky side chains yielded poor substrate specificity. The k cat /K M values for peptides with P 2 ′ substitutions containing Tyr in P 3 were higher than those obtained for the peptides without Tyr. In fact, P′ and P modifications mainly promoted changes in k cat and K M , respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a polypeptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism.