Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease

Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):922-6. doi: 10.1073/pnas.89.3.922.


The prohormone-processing Kex2 protease of the budding yeast Saccharomyces cerevisiae can be converted from an intracellular membrane protein to a soluble, secreted, and active form by deletion of the transmembrane domain and C-terminal tail. One such molecule was purified to near homogeneity from the culture medium of an overexpressing yeast strain. Amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially autoproteolytic cleavage at Lys108-Arg109, prior to the domain homologous to subtilisin, followed by trimming of Leu-Pro and Val-Pro dipeptides by the Ste13 dipeptidyl aminopeptidase. Kinetic parameters were examined using fluorogenic peptidyl-methylcoumarin amide substrates. Initial burst titration indicated that the preparation was entirely active. Measurements of dependence of activity on pH yielded a simple curve suggesting titration of a single ionizable group. Activity was half-maximal at pH 5.7 and nearly constant from pH 6.5 to 9.5. Discrimination between substrates was as great as 360-fold in Km and 130-fold in kcat. Substrates with a Lys-Arg dipeptide preceding the cleaved bond were preferred, having kcat/Km values up to 1.1 x 10(7) sec-1.M-1. The enzyme cleaved substrates having Arg-Arg, Pro-Arg, Ala-Arg, and Thr-Arg with increased Km but with unchanged kcat. In contrast, the enzyme displayed a dramatically lower kcat for a Lys-Lys substrate with a smaller increase in Km. Thus the two residues preceding the cleaved bond may play distinct roles in the selectivity of binding and cleavage of prohormone substrates.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • DNA Mutational Analysis
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Glycoproteins / chemistry
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Peptides / metabolism
  • Proprotein Convertases*
  • Protein Precursors / metabolism*
  • Recombinant Proteins
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae Proteins*
  • Serine Endopeptidases / chemistry*
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism
  • Solubility
  • Structure-Activity Relationship
  • Substrate Specificity
  • Subtilisins*


  • Fungal Proteins
  • Glycoproteins
  • Peptides
  • Protein Precursors
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Proprotein Convertases
  • Serine Endopeptidases
  • Subtilisins
  • KEX2 protein, S cerevisiae