STABILIZATION OF LYSOZYME AGAINST IRREVERSIBLE INACTIVATION BY ALTERATIONS OF THE ASP-GLY SEQUENCES

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 prot ect the enzyme against irreversible thermoinactivation, Because the ly sozyme inactivation was caused by the accumulation of multiple chemica l reactions, including the isomerization of the Asp-Gly sequence and t he 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 rep laced) were prepared, The substitution resulted in the reversible dest abilization from 1 to 2 kcal/ mol per substitution, The destabilizatio n was caused by the introduction of beta-carbon to the constrained pos ition that had conformational angles within the allowed range for the Gly residue, Despite the decrease in the reversible conformational sta bility, the mutants had more resistance to irreversible inactivation a t pH 4 and 100 degrees C, In particular, the rate of irreversible inac tivation of the mutant, which was replaced at four chemically labile s equences, was the latest and corresponded to similar to 18 kcal/mol of the reversible conformational stability, Therefore, replacement of th e chemically labile sequence was found to be more effective at protect ing enzymes against irreversible thermoinactivation than at strengthen ing reversible conformational stability.