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
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:41 p.m.
|Number of data points||15|
|Proteins||Lysozyme C ; Lysozyme C|
|Assays/Quantities/Protocols||Experimental Assay: activity ; Experimental Assay: Cm ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL|