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 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.
Nutritional Science Laboratory, Morinaga Milk Industry Company Ltd., 5-1-83 Higashihara, Zama, Kanagawa, 228-8583, Japan.
K
K
Shin
H
H
Hayasawa
B
B
Lönnerdal
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D006801:Humans; D015151:Immunoblotting; D007784:Lactoperoxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D007784:Lactoperoxidase; D009195:Peroxidase; D002118:Calcium