Mutations affecting the calcium-binding site of myeloperoxidase and lactoperoxidase

Both myeloperoxidase (MPO) and lactoperoxidase (LPO) contain high affinity bound calcium, which has been suggested to play a structural role. Asp-96 i n MPO, a residue next to the histidine distal from the heme prosthetic grou p, has been assigned to the calcium-binding site of the enzyme by X-ray cry stallography. Multiple sequence alignment of known animal peroxidases has r evealed that the calcium-binding site is highly conserved. In this study, w e replaced Asp-96 in MPO and the counterpart Asp-227 in LPO both with Ala b y site-directed mutagenesis. The level of peroxidase activity in insect cel ls infected with recombinant baculoviruses and their culture supernatants w as 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 tha t a functional calcium-binding site is essential for the biosynthesis of ac tive animal peroxidases. (C) 2001 Acaaemic Press.