Mutations affecting the calcium-binding site of myeloperoxidase and lactoperoxidase

Kouichirou Shin, Hirotoshi Hayasawa, Bo Lönnerdal

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Both myeloperoxidase (MPO) and lactoperoxidase (LPO) contain high affinity bound calcium, which has been suggested to play a structural role. Asp-96 in MPO, a residue next to the histidine distal from the heme prosthetic group, has been assigned to the calcium-binding site of the enzyme by X-ray crystallography. Multiple sequence alignment of known animal peroxidases has revealed that the calcium-binding site is highly conserved. In this study, we replaced Asp-96 in MPO and the counterpart Asp-227 in LPO both with Ala by site-directed mutagenesis. The level of peroxidase activity in insect cells infected with recombinant baculoviruses and their culture supernatants was reduced to virtually zero as a result of these mutations. Immunoblotting revealed that these mutant peroxidases were expressed in the cells but not secreted as effectively as the wild-type enzymes. Our findings suggest that a functional calcium-binding site is essential for the biosynthesis of active animal peroxidases.

Original languageEnglish (US)
Pages (from-to)1024-1029
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume281
Issue number4
DOIs
StatePublished - 2001

Keywords

  • Baculovirus-insect cell system
  • Calcium-binding site
  • Lactoperoxidase
  • Myeloperoxidase
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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