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 peptidylglycin e alpha-amidating monooxygenase (PAM) protein. Tissue-specific alterna tive splicing and endoproteolysis are known to generate both soluble a nd integral membrane mono- and bifunctional PAM proteins. In order to investigate the functional consequences of these differences we purifi ed PAM-3, a soluble 95-kDa bifunctional form of the enzyme, from the s pent medium of stably transfected hEK-293 cells. Using NH2-terminal se quence analysis of products of limited endoproteolysis and antibody cr oss-reactivity we identified protease-sensitive regions at the NH2 ter minus, between the 35-kDa PHM and 42-kDa PAL domains and at the COOH t erminus of the protein. Endoproteolytic removal of the COOH-terminal r egion from the bifunctional PAM-3 protein shifted the pH optimum of PH M to a more alkaline pH, increased the turnover number (k(cat)) of PHM and decreased its K(M) for alpha-N-acetyl-Tyr-Val-Gly; the catalytic properties of PAL were not altered. Since peptide amidation can be a r ate-limiting step in the biosynthesis of neuropeptides, similar increa ses in PHM activity in vivo may play an important role in regulating t he extent of peptide alpha-amidation.