ONE-STEP SITE-DIRECTED MUTAGENESIS OF THE KEX2 PROTEASE OXYANION HOLE

Background: Members of the subtilisin family of serine proteases usual ly have a conserved asparagine residue that stabilizes the oxyanion tr ansition state of peptide-bond hydrolysis. Yeast Kex2 protease is a me mber of the subtilisin family that differs from die degradative subtil isin proteases in its high substrate specificity; it processes pro-alp ha-factor, the precursor of the alpha-factor mating pheromone of yeast , and also removes the propeptide from its own precursor by an intramo lecular cleavage reaction. Curiously, the mammalian protease PC2, a Ke x2 homolog that is likely to be required for pro-insulin processing, h as an aspartate in place of asparagine at the 'oxyanion hole'. Results : We have tested the effect of making substitutions of the conserved o xyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in ye ast. Using this method, we have substituted alanine or aspartate for A sn 314 in a form of Kex2 engineered for secretion. Transformants expre ssing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstabl e but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vit ro. We found that, with three peptides that are good substrates of wil d-type Kex2, the k(cat) of the Asp 314 enzyme was reduced approximatel y 4500-fold and its K(M) approximately 4-fold, relative to the wild-ty pe enzyme. For the peptide substrate corresponding to the cleavage sit e of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduc ed only 125-fold, while the K(M) was increased 3-fold. Despite its red uced catalytic activity, however, processing of die mutant enzyme in v ivo - by the intramolecular cleavage that removes its amino-terminal p ro-domain - occurs at an unchanged rate. Conclusions: The effects of t he Asn 314-Asp substitution reveal contributions to die reaction speci ficity of the Kex2 protease of substrate residues amino-terminal to th e pair of basic residues at the cleavage site. Aspartate at the oxyani on hole appears to confer k(cat)discrimination between substrates by r aising the energy barrier for productive substrate binding: this may h ave implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecul ar cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding.