Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences.


Abstract

Site-directed mutagenesis was performed at Asp-Gly (48-49, 66-67, 101-102) and Asn-Gly (103-104) sequences of hen egg-white lysozyme to protect the enzyme against irreversible thermoinactivation. Because the lysozyme inactivation was caused by the accumulation of multiple chemical reactions, including the isomerization of the Asp-Gly sequence and the deamidation of Asn [Tomizawa et al. (1994) Biochemistry, 33, 13032-13037], the suppression of these reactions by the substitution of Gly to Ala, or the introduction of a sequence of human-type lysozyme, was attempted and the mutants (where each or all labile sequences were replaced) were prepared. The substitution resulted in the reversible destabilization from 1 to 2 kcal/mol per substitution. The destabilization was caused by the introduction of beta-carbon to the constrained position that had conformational angles within the allowed range for the Gly residue. Despite the decrease in the reversible conformational stability, the mutants had more resistance to irreversible inactivation at pH 4 and 100 degrees C. In particular, the rate of irreversible inactivation of the mutant, which was replaced at four chemically labile sequences, was the latest and corresponded to approximately 18 kcal/mol of the reversible conformational stability. Therefore, replacement of the chemically labile sequence was found to be more effective at protecting enzymes against irreversible thermoinactivation than at strengthening reversible conformational stability. Study holds ProTherm entries: 11245, 11246, 11247, 11248 Extra Details: aspartic isomerization; inactivation; lysozyme;,mutagenesis; stabilization

Submission Details

ID: pkJifc2X3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Tomizawa H;Yamada H;Hashimoto Y;Imoto T,Protein Eng. (1995) Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences. PMID:8771183
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2IHL 1993-06-29T00:00:00+0000 1.4 LYSOZYME (E.C.3.2.1.17) (JAPANESE QUAIL)
1FBI 1995-01-19T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF A CROSS-REACTION COMPLEX BETWEEN FAB F9.13.7 AND GUINEA-FOWL LYSOZYME
1GHL 1993-05-04T00:00:00+0000 2.1 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1HHL 1993-05-04T00:00:00+0000 1.9 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1JHL 1993-05-04T00:00:00+0000 2.4 THREE-DIMENSIONAL STRUCTURE OF A HETEROCLITIC ANTIGEN-ANTIBODY CROSS-REACTION COMPLEX
1BQL 1995-02-03T00:00:00+0000 2.6 STRUCTURE OF AN ANTI-HEL FAB FRAGMENT COMPLEXED WITH BOBWHITE QUAIL LYSOZYME
1DKJ 1996-01-10T00:00:00+0000 2.0 BOBWHITE QUAIL LYSOZYME
1DKK 1996-01-10T00:00:00+0000 1.9 BOBWHITE QUAIL LYSOZYME WITH NITRATE
135L 1993-06-10T00:00:00+0000 1.3 X-RAY STRUCTURE OF MONOCLINIC TURKEY EGG LYSOZYME AT 1.3 ANGSTROMS RESOLUTION
1DZB 2000-02-23T00:00:00+0000 2.0 Crystal structure of phage library-derived single-chain Fv fragment 1F9 in complex with turkey egg-white lysozyme

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.3 Lysozyme C P49663 LYSC_PHAVE
93.0 Lysozyme C P81711 LYSC_SYRSO
93.2 Lysozyme C P00702 LYSC_PHACO
94.6 Lysozyme C P24533 LYSC_SYRRE
93.0 Lysozyme C P24364 LYSC_LOPLE
92.2 Lysozyme C P00704 LYSC_NUMME
95.2 Lysozyme C P00703 LYSC_MELGA
95.3 Lysozyme C Q7LZT2 LYSC_TRATE
95.3 Lysozyme C P22910 LYSC_CHRAM
96.1 Lysozyme C P19849 LYSC_PAVCR
95.3 Lysozyme C P00701 LYSC_COTJA
96.1 Lysozyme C Q7LZI3 LYSC_TRASA
96.9 Lysozyme C Q7LZP9 LYSC_LOPIM
96.9 Lysozyme C Q7LZQ0 LYSC_CATWA
96.9 Lysozyme C P00699 LYSC_CALCC
96.9 Lysozyme C P00700 LYSC_COLVI
100.0 Lysozyme C P00698 LYSC_CHICK