Thermodynamic analysis of Kex2 activity: The acylation and deacylation steps are potassium- and substrate-dependent.

Kex2 is the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave at sites containing pairs of basic residues. Here, we studied the effects of KCl on the individual rate constants of association, dissociation, acylation and deacylation and determined the thermodynamic parameters at each step of the Kex2 reaction. Potassium bound Kex2 with KD=20.3mM. The order in which potassium entered the reaction system modified the effect of activation or inhibition, which depended on the size of the substrate. A possible allosteric potassium binding site at the S6 subsite was involved in activation, and a distant site located between the catalytic domain and the P-domain was involved in inhibition. Potassium decreased the energetic barriers of almost all steps of catalysis. The acylation of Ac-PMYKR-AMC in the absence of potassium was the rate-limiting step. Therefore, for substrates containing a P1-Arg, the deacylation step is not necessarily the rate-limiting event, and other residues at the P' positions may participate in controlling the acylation and deacylation steps. Thus, it is reasonable to conclude that potassium is involved in the processing of the α-mating factor that promotes Ca2+ mobilization by activating a high-affinity Ca2+-influx system to increase the cytosolic [Ca2+], resulting in the activation of channels that are essential for the survival of Saccharomyces cerevisiae cells.

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

Kex2 is a Ca2+-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 P1' is affected by the type of residue in the P3 position. Some residues in P1' with large or bulky side chains yielded poor substrate specificity. The kcat/KM values for peptides with P2' substitutions containing Tyr in P3 were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in kcat and KM, 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.