Reaction of Calcium-activated Neutral Protease (CANP) with an Epoxysuccinyl Derivative (E64c) and Iodoacetic Acid

The reaction of calcium activated neutral protease (CANP) with an epoxysuccinyl derivative (E64c) and iodoacetic acid (IAA) was examined in the presence of Ca2+, which specifically accelerates the rate of inactivation by these inhibitors. 1. E64c and IAA (100-fold molar excess) each inactivated CANP within a few minutes at 0 degrees C. Upon inactivation, 1 mol of E64c or IAA was incorporated into CANP. The reaction of CANP with E64c or IAA was optimal at pH 7.5-8.0, where CANP shows the maximum enzyme activity. 2. CANP modified with E64c or IAA lost one of the 3 SH groups (class II SH groups) which were exposed at the surface by addition of Ca2+. No conformation change of CANP was observed as a result of the modifications. 3. Leupeptin inhibited the modifications. A total of 1 mol of E64c and IAA was incorporated into CANP by successive labeling of CANP with E64c and IAA. It was concluded from these results that E64c and IAA specifically react with the same class II SH group, which is regarded as the active site of CANP.