Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression.
The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.
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Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Perkins
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Husten
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Johnson
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Keutmann
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Mains
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D019760:Amidine-Lyases; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002413:Cations, Divalent; D002417:Cattle; D003594:Cytoplasmic Granules / Q000201:enzymology; D004247:DNA / Q000235:genetics; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000201:enzymology*
D002413:Cations, Divalent; D008670:Metals; D004247:DNA; D004492:Edetic Acid; D006836:Hydro-Lyases; D019760:Amidine-Lyases; C068521:peptidylamidoglycolate lyase