Mutations affecting the calcium-binding site of myeloperoxidase and lactoperoxidase |
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Authors: | Shin K Hayasawa H Lönnerdal B |
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Affiliation: | Nutritional Science Laboratory, Morinaga Milk Industry Company Ltd., 5-1-83 Higashihara, Zama, Kanagawa, 228-8583, Japan. |
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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. |
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