Reaction of neutral endopeptidase 24.11 (enkephalinase) with arginine reagents

The effect of the arginine-specific reagents phenylglyoxal and butanedione on the activity of neutral endopeptidase 24.11 ('enkephalinase') was determined. Inactivation of the enzyme by butanedione is completely protected by methionine-enkephalin, but only partially protected by phenylglyoxal amide. In contrast, phenylglyoxal inactivation of the enzyme exhibits saturation kinetics with a K(d) of 20 mM. The enzyme is only partially protected against phenylglyoxal inactivation by both methionine-enkephalin and its amide, indicating that phenylglyoxal reacts at two sites. Reaction of the enzyme with phenylglyoxal in the presence of saturating methionine-enkephalin involves the direct reaction of the reagent with the enzyme-substrate complex. Enzyme treated with butanedione or with phenylglyoxal (at site 1) exhibits a 3-5 decrease in substrate binding with little change in k(cat). In contrast, reaction with phenylglyoxal in the presence of saturating methionine-enkephalin shows little change in substrate binding but a 4-fold decrease in k(cat). Enzyme inactivation involves the incorporation of approximately 1 mol of phenylglyoxal/enzyme subunit in the absence of methionine-enkephalin and approximately 2.5 mol of phenylglyoxal/enzyme subunit in the presence of saturating methionine-enkephalin. These results suggest that an arginine residue on the enzyme is involved in substrate binding.