Characterization and kinetic analysis of enzyme-substrate recognition by three recombinant lactococcal tripeptidases

Biochim Biophys Acta. 2005 Apr 15;1748(1):26-34. doi: 10.1016/j.bbapap.2004.12.001. Epub 2005 Jan 21.


Tripeptidases from Lactococcus lactis subsp. lactis (L9PepTR), L. lactis subsp. cremoris (L6PepTR), and L. lactis subsp. hordniae (hTPepTR) were cloned, overexpressed, purified, and characterized. Although these enzymes contained three to seven naturally occurring amino acid differences, both metal-binding and catalytic sites were highly conserved. The k(cat) values of hTPepTR were approximately 1.5- to 2-fold higher than those of L9PepTR, while, for L6PepTR, they were approximately 0.8- to 1.4-times the L9PepTR values. The K(m) of tripeptidase from subsp. lactis (L9PepTR) was considerably larger when glycine was the amino acid located at both the N- and C-terminus of the peptide substrate. In addition, the K(m) values of L9PepTR increased in the following order for YGG, LGG, FGG, SGG, and alpha-aminoisobutyrylglycylglycine, while the k(cat)/K(m) decreased in the same order. These results suggest that the dipole moment and steric hindrance of the N-terminal amino acid side chain may be the most important factors controlling substrate specificity.

MeSH terms

  • Amino Acid Sequence
  • Aminopeptidases / chemistry
  • Aminopeptidases / genetics
  • Aminopeptidases / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Hydrogen-Ion Concentration
  • Lactococcus lactis / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Sequence Alignment
  • Substrate Specificity
  • Temperature


  • Bacterial Proteins
  • Recombinant Proteins
  • Aminopeptidases
  • PepT tripeptidase