Publications

• Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2.

  Han M., Park D., Vanderzalm P.J., Mains R.E., Eipper B.A., Taghert P.H.

  Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycin ... >> More

  Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo. Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K(m) values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes. << Less

  J. Neurochem. 90:129-141(2004) [PubMed] [EuropePMC]

• A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation.

  Katopodis A.G., Ping D., May S.W.

  We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa an ... >> More

  We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD. << Less

  Biochemistry 29:6115-6120(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme.

  Bell J., Ash D.E., Snyder L.M., Kulathila R., Blackburn N.J., Merkler D.J.

  Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the alpha-carbon of the glycyl ... >> More

  Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the alpha-carbon of the glycyl residue, producing an alpha-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that alpha-AE requires 1.1 +/-0. 2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-alpha-hydroxyglycine dealkylation activity. Treatment of the enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restoration of dealkylation activity, only 3% relative to an untreated control. The structure and coordination of the zinc center has been investigated by X-ray absorption spectroscopy. EXAFS data are best interpreted by an average coordination of 2-3 histidine ligands and 1-2 non-histidine O/N ligands. Since catalytic zinc centers in other zinc metalloenzymes generally exhibit only O/N ligands to the zinc atom, a zinc-bound water or hydroxide may serve as a general base for the abstraction of the hydroxyl proton from the carbinolamide intermediate. Alternatively, the zinc may function in a structural role. << Less

  Biochemistry 36:16239-16246(1997) [PubMed] [EuropePMC]