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

Arch Biochem Biophys. 2006 Oct 15;454(2):137-45. doi: 10.1016/j.abb.2006.08.001. Epub 2006 Aug 22.

Abstract

The dipeptidases (PepVs) from three typical lactococcal strains, Lactococcus lactis subsp. lactis (L9), L. lactis subsp. cremoris (L6) and L. lactis subsp. hordniae (hT) were cloned and characterized. The metal-binding, catalytic, and substrate-binding sites are highly conserved among of them. A computer-generated three-dimensional model suggested that the amino acid differences between these PepVs were mostly located away from the active center. L9 PepV does not hydrolyze dipeptides bearing Pro or D-amino acid at the C-terminal amino acid. Unlike PepV from Lactobacillus delbrueckii, L9 PepV does not cleave beta-Asp-His, and has little ability to cleave dipeptides containing a beta-alanine. In addition, L9 PepV has a much higher kcat for dipeptides with an N-terminal Ala but a significantly higher Km when the N-terminal amino acid is Gly. The substrate recognition profile of PepV is further discussed on the basis of the kinetic analysis and the structural model.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Dipeptidases / chemistry
  • Dipeptidases / isolation & purification
  • Dipeptidases / metabolism*
  • Dipeptides / metabolism*
  • Electrophoresis, Capillary
  • Kinetics
  • Lactococcus lactis / enzymology*
  • Models, Molecular
  • Molecular Sequence Data
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Dipeptides
  • Recombinant Proteins
  • Dipeptidases