Use of endoproteases to identify catalytic domains, linker regions, and functional interactions in soluble peptidylglycine alpha-amidating monooxygenase

• Published in: The Journal of biological chemistry Vol. 268; no. 13; pp. 9709 - 9717
• Main Authors: HUSTEN, E. J, TAUSK, F. A, KEUTMANN, H. T, EIPPER, B. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.05.1993
• Subjects: Alternative Splicing; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blotting, Western; Cell Line; cell lines; Electrophoresis, Polyacrylamide Gel; Endopeptidases - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Kidney; Kinetics; man; Metalloendopeptidases; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - isolation & purification; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Multienzyme Complexes; Oxidoreductases; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; peptidylglycine alpha -amidating monooxygenase; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; structure-activity relationships; Transfection; Trypsin; Human; Enzymatic activity; Structure activity relation; Enzyme; Peptidylglycine monooxygenase; Catalytic site; Kinetics; Amidation; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)98406-1

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.