Selective modification of aspartic acid-101 in lysozyme by carbodiimide reaction

Abstract

A general procedure which selectively introduced a nucleophilic group at a particular location in the active site of lysozyme has been developed. The coupling of hen egg white lysozyme with amine nucleophiles by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was studied at pH 5 and room temperature. In the presence of an amine nucleophile, such as ethanolamine, ethylenediamine, methylamine, or 4(5)-(aminomethyl)imidazole, the carboxyl side chain of aspartic acid-101 in lysozyme was selectively modified by using a small excess of EDC. The reactivity of Asp-101 is probably due to the specific binding of EDC to the substrate binding site close to Asp-101. With histamine or D-glucosamine, the selectively of Asp-101 was somewhat decreased. This may be due to the specific binding of these amines to lysozyme in competition with EDC, causing a decrease of the selective activation of Asp-101 by EDC. Depending on the amine employed, the lysozyme derivatives obtained exhibited decreased activity (83-52% of native enzyme), suggesting that the modification of Asp-101 weakened substrate binding.