Identification of Aspartic Acid 52 as the Point of Attachment of an Affinity Label in Hen Egg White Lysozyme

Hen egg white lysozyme, fully inactivated by the affinity-labeling reagent, the 2',3'-epoxypropyl β-glycoside of di-(N-acetyl-d-glucosamine), contained 1 mole of the label per mole of protein. Total enzymatic digestion of the reduced and carboxymethylated affinity-labeled enzyme afforded, as a major radioactive product, a compound composed solely of aspartic acid and glucosamine at the molar ratio of 1:2. Peptic digestion afforded a single radioactive peptide corresponding to the sequence [see PDF for sequence] in the enzyme. Digestion of this peptide with Clostridium histolyticum aminopeptidase gave a radioactive pentapeptide the composition, partial structure, and other properties of which corresponded to the sequence [see PDF for sequence] in hen egg white lysozyme, and which contained 2 moles of glucosamine per mole of peptide. Our findings show that the affinity label is covalently bound to the enzyme via the β-carboxyl group of Asp 52 and are in accord with our conclusions from immunochemical (Maron, E., Eshdat, Y., and Sharon, N. (1972) Biochim. Biophys. Acta 278, 243) and x-ray crystallographic (Moult, J., Eshdat, Y., and Sharon, N. (1973) J. Mol. Biol. 75, 1) studies of the affinity-labeled hen egg white lysozyme.