A biochemical analysis of substrate specificity for the <italic>Saccharomyces cerevisiae</italic> endoprotease, Kex2, and the Kex2-related Prt1 protease from <italic>Pneumocystis carinii</italic>.

Abstract

Many essential biological functions, including apoptosis, blood coagulation, and hormonal signaling, are regulated, in part, by proteolysis. To serve as a means of regulation, proteolytic cleavage must be performed in an extremely specific fashion because aberrant cleavage of non-substrate proteins would have unwanted consequences. Members of the Kex2/furin family of processing proteases cleaves a variety of substrates in the eukaryotic secretory pathway. Although this family of proteases contain the same catalytic fold and similarly hydrolyze peptide bonds C-terminal to basic residues, the consensus cleavage sites of these well characterized proteases are distinct from one another. The substrate specificity of the prototypical member of this family, Kex2 from <italic>S. cerevisiae</italic>, was investigated using several strategies. First, the substrate specificity for P₄ and P₆ residues was analyzed by substituting basic residues into the putative P₄ and P₆ binding pockets by site directed mutagenesis. Whereas Kex2 shows little recognition for P₆ Arg, one mutant, T252D Q283E-Kex2, exhibited 15-fold preference for Arg versus Ala at P₆. Another mutant, E255I-Kex2 was predicted to reduce recognition for Arg at P4, and as expected, it showed decreased processing of P₄ Arg peptide substrates in some contexts. In a second set of experiments, a series of Kex2 mutants with altered P₂ specificity that were obtained in a genetic screen were analyzed biochemically. The processing of wild-type and the target specificity was less dramatic than observed in the genetic assay. However, they followed the same general trends in improved cleavage of non-native P₂ substrates. Also, an attempt was made to redesign a genetic selection to obtain more robust Kex2 mutants with arbitrary P₂ specificity, but this effort was hampered by a prohibitively high background. Finally, a novel member of the Kex2/furin family, Prt1 from the fungal pathogen <italic>P. carinii</italic>, was expressed in Sf9 cells and purified. N-terminal sequencing data indicated that the propeptide was not autocatalytically processed. It is possible that this protease needs an activating protein that is not expressed in insect cells or more physiological conditions for autoproteolysis of its propeptide.