QUANTITATIVE ASSESSMENT OF ENZYME SPECIFICITY IN-VIVO - P-2 RECOGNITION BY KEX2 PROTEASE DEFINED IN A GENETIC SYSTEM

The specificity of the yeast proprotein-processing Ke2 protease was ex amined in vivo by using a sensitive, quantitative assay. A truncated p repro-cu-factor gene encoding an cy-factor precursor with a single cu- factor repeat was constructed with restriction sites for cassette muta genesis flanking the single Kex2 cleavage site (-SLDKR down arrow EAEA -), All of the 19 substitutions for the Lys (Pz) residue in the cleava ge site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prep ro-alpha-factor, Cleavage of the 20 sites by Kex2, expressed at the wi ld-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitut ions for Lys at P-2 decreased mating, from 2-fold for Arg to >10(6)-fo ld for Trp, Eviction of the Kex2-encoding plasmid indicated that cleav age of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precurso rs correlated well with the specificity (k(cat)/K-M) of purified Kex2 for comparable model peptide substrates, validating the in vivo approa ch as a quantitative method. The results support the conclusion that K -M, which is heavily influenced by the nature of the P-2 residue, is a major determinant of cleavage efficiency in vivo. P-2 preference foll owed the rank order: Lys > Arg > Thr > Pro > Glu > tie > Ser > Ala > A sn > Val > Cys > AsP > Gin > Gly > His > Met > Leu > Tyr > Phe > Trp.