Use of endoproteases to identify catalytic domains, linker regions, and functional interactions in soluble peptidylglycine alpha-amidating monooxygenase.
The production of alpha-amidated peptides is accomplished through the sequential action of two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), that are contained within the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein. Tissue-specific alternative splicing and endoproteolysis are known to generate both soluble and integral membrane mono- and bifunctional PAM proteins. In order to investigate the functional consequences of these differences we purified PAM-3, a soluble 95-kDa bifunctional form of the enzyme, from the spent medium of stably transfected hEK-293 cells. Using NH2-terminal sequence analysis of products of limited endoproteolysis and antibody cross-reactivity we identified protease-sensitive regions at the NH2 terminus, between the 35-kDa PHM and 42-kDa PAL domains and at the COOH terminus of the protein. Endoproteolytic removal of the COOH-terminal region from the bifunctional PAM-3 protein shifted the pH optimum of PHM to a more alkaline pH, increased the turnover number (kappa(cat)) of PHM and decreased its KM for alpha-N-acetyl-Tyr-Val-Gly; the catalytic properties of PAL were not altered. Since peptide amidation can be a rate-limiting step in the biosynthesis of neuropeptides, similar increases in PHM activity in vivo may play an important role in regulating the extent of peptide alpha-amidation.
Husten
E J
EJ
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Tausk
F A
FA
Keutmann
H T
HT
Eipper
B A
BA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008666:Metalloendopeptidases; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014357:Trypsin
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase