Selective modification of aspartic acid-101 in lysozyme by carbodiimide reaction.
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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002645:Chickens; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004529:Egg White; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005260:Female; D009113:Muramidase / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding
D000596:Amino Acids; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D001224:Aspartic Acid; D009113:Muramidase; D005022:Ethyldimethylaminopropyl Carbodiimide