Peptide amidation in an invertebrate: Purification, characterization, and inhibition of peptidylglycine α‐hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

Peptidylglycine α‐hydroxylating monooxygenase (PHM), an enzyme involved in formation of neuropeptides with a C‐terminal amide functionality in mammals and amphibians, was isolated from the head of an invertebrate, the honeybee, Apis mellifera, and purified 220‐fold in 1% overall yield. The bee PHM has a molecular weight of 71,000, is membrane associated but can be solubilized with a detergent (n‐octyl‐β‐D‐glucopyranoside), 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 dansyl‐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 μM and the V_max is 2.3 nmol • μg⁻¹ • h⁻¹. Treatment of the insect PHM with D‐Phe‐L‐Phe‐D‐vinylglycine, a substrate analogue and mechanism‐based inactivator of PHM from pig pituitary, results in irreversible loss of activity. The diastereomeric analogue, D‐Phe‐L‐Phe‐L‐vinylglycine, is only a competitive inhibitor (lC₅₀ = 320 μM). © 1994 Wiley‐Liss, Inc.