title: Design and creation of a Ca2+ binding site in human lysozyme to enhance structural stability.
abstract: A Ca2+ binding site like an EF-hand motif was designed and created in human lysozyme by replacing both Gln-86 and Ala-92 with aspartic acids by site-directed mutagenesis. The mutant human lysozyme (D86/92-lysozyme) was expressed and secreted by yeast. One Ca2+ was found to bind one molecule of the purified protein with the binding constant 5.0 x 10(6) M-1. The enzymatic activity of holo-D86/92-lysozyme against glycol chitin at 40 degrees C was 2-fold higher than that of the native lysozyme. Maximal activity of the holo-D86/92-lysozyme was observed at 80 degrees C, where its relative activity normalized to the value at 40 degrees C was 6-fold and 17-fold higher than those of the native and apoenzymes, respectively. The activities of the native lysozyme and apo-D86/92-lysozyme were maximum at 65 degrees C-70 degrees C. Moreover, D86/92-lysozyme was more stable against protease digestion than the native lysozyme. These results indicate that the creation of the calcium binding site like an EF-hand motif in the human lysozyme enhances its structural stability.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Kuroki
authors.forename: Y
authors.initials: Y
authors.lastname: Taniyama
authors.forename: C
authors.initials: C
authors.lastname: Seko
authors.forename: H
authors.initials: H
authors.lastname: Nakamura
authors.forename: M
authors.initials: M
authors.lastname: Kikuchi
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013816:Thermodynamics
chemical_list: D004247:DNA; D009113:Muramidase; D002118:Calcium