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 methionine-enkephalinamide. In contrast, phenylglyoxal inactivation of the enzyme exhibits saturation kinetics with a Kd 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 kcat. In contrast, reaction with phenylglyoxal in the presence of saturating methionine-enkephalin shows little change in substrate binding but a 4-fold decrease in kcat. 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.
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001120:Arginine; D002074:Butanones / Q000494:pharmacology*; D003931:Diacetyl / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism*; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D015260:Neprilysin; D010658:Phenylglyoxal / Q000494:pharmacology*; D011480:Protease Inhibitors; D051381:Rats
D000447:Aldehydes; D002074:Butanones; D011480:Protease Inhibitors; D004744:Enkephalin, Methionine; C021055:Met-enkephalinamide; D001120:Arginine; D010450:Endopeptidases; D015260:Neprilysin; D003931:Diacetyl; D010658:Phenylglyoxal