Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10384-9. doi: 10.1073/pnas.95.18.10384.


The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-alpha-factor gene encoding an alpha-factor precursor with a single alpha-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR downward arrowEAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-alpha-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >10(6)-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Hydrolysis
  • Lysine / metabolism*
  • Mutagenesis, Insertional
  • Proprotein Convertases*
  • Saccharomyces cerevisiae Proteins*
  • Sensitivity and Specificity
  • Substrate Specificity
  • Subtilisins / genetics
  • Subtilisins / metabolism*


  • Saccharomyces cerevisiae Proteins
  • Proprotein Convertases
  • Subtilisins
  • KEX2 protein, S cerevisiae
  • Lysine