Peptide amidation in an invertebrate: purification, characterization, and inhibition of peptidylglycine alpha-hydroxylating monooxygenase from the heads of honeybees (Apis mellifera).
Peptidylglycine alpha-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-beta-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 Km is 1.7 microM and the Vmax is 2.3 nmol.micrograms-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 (IC50 = 320 microM).
Zabriskie
T M
TM
Department of Chemistry, University of Alberta, Edmonton, Canada.
Klinge
M
M
Szymanski
C M
CM
Cheng
H
H
Vederas
J C
JC
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees / Q000378:metabolism*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009097:Multienzyme Complexes; D013379:Substrate Specificity
D000577:Amides; D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase