PEPTIDE AMIDATION IN AN INVERTEBRATE - PURIFICATION, CHARACTERIZATIONAND INHIBITION OF PEPTIDYLGLYCINE ALPHA-HYDROXYLATING MONOOXYGENASE FROM THE HEADS OF HONEYBEES (APIS-MELLIFERA)

Peptidylglycine alpha-hydroxylating monooxygenase (PHM), an enzyme inv olved in formation of neuropeptides with a C-terminal amide functional ity in mammals and amphibians, was isolated from the head of an invert ebrate, the honeybee, Apis mellifera, and purified 220-fold in 1% over all yield. The bee PHM has a molecular weight of 71,000, is membrane a ssociated but can be solubilized with a detergent (n-octyl-beta-D-gluc opyranoside), and cross-reacts with rabbit antibodies generated toward bacterially expressed rat PHM. In the presence of copper, oxygen, and ascorbic acid, the enzyme hydroxylates model tripeptides such as dans yl-L-Phe-L-Phe-Gly on the methylene carbon of the glycine residue with retention of configuration. Using this tripeptide as substrate, the K -m is 1.7 mu M and the V-max is 2.3 nmol.mu g(-1) h(-1). Treatment of the insect PHM with D-Phe-L-Phe-D-vinylglycine, a substrate analogue a nd mechanism-based inactivator of PHM from pig pituitary, results in i rreversible loss of activity. The diastereomeric analogue, D-Phe-L-Phe -L-vinylglycine, is only a competitive inhibitor (IC50 = 320 mu M). (C ) 1994 Wiley-Liss, Inc.