| Literature DB >> 2674939 |
R Kuroki1, Y Taniyama, C Seko, H Nakamura, M Kikuchi, M Ikehara.
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.Entities:
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Year: 1989 PMID： 2674939 PMCID： PMC297958 DOI： 10.1073/pnas.86.18.6903
Source DB: PubMed Journal: Proc Natl Acad Sci U S A ISSN： 0027-8424 Impact factor: 11.205