Peptidylglycine-alpha-hydroxylating monooxygenase
The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (Phm) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic domains necessary for the two-step formation of alpha-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA have been identified by examining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contains exon A, the 315-nucleotide (nt) segment situated between the Phm and PAL domains and present in rPAM-1 but absent from rPAM-2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment encoding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane protein; rPAM-3a lacks a 204-nt segment, including the transmembrane domain, and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the Phm domain; alternative splicing generates a novel 3'-region encoding a COOH-terminal pentapeptide followed by 1.1 kb of 3'-untranslated region. The soluble rPAM-5 protein lacks PAL, transmembrane, and cytoplasmic domains. These three forms of PAM mRNA can be generated by alternative splicing. The major forms of PAM mRNA in both lobes of the pituitary are rPAM-3b and rPAM-2. Despite the fact that anterior and neurointermediate pituitary contain a similar distribution of forms of PAM mRNA, the distribution of PAM proteins in the two lobes of the pituitary is quite different. Although integral membrane proteins similar to rPAM-2 and rPAM-3b are major components of anterior pituitary granules, the PAM proteins in the neurointermediate lobe have undergone more extensive endoproteolytic processing, and a 75-kDa protein containing both Phm and PAL domains predominates. The bifunctional PAM precursor undergoes tissue-specific endoproteolytic cleavage reminiscent of the processing of prohormones (Eipper, 1992).
Peptidylglycine alpha-hydroxylating monooxygenase (Phm) is a copper, ascorbate, and molecular oxygen dependent enzyme that plays a key role in the biosynthesis of many peptides. Using site-directed mutagenesis, the catalytic core of Phm was found not to extend beyond Asp359. Shorter Phm proteins are eliminated intracellularly, suggesting that they fail to fold correctly. A set of mutant Phm proteins whose design is based on the structural and mechanistic similarities of Phm and dopamine beta-monooxygenase (D beta M) was characterized. Mutation of Tyr79, the residue equivalent to a p-cresol target in D beta M, to Phe79 alters the kinetic parameters of Phm. Disruption of either His-rich cluster contained within the Phm/D beta M homology domain eliminates activity, while deletion of a third His-rich cluster unique to Phm fails to affect activity; the catalytically inactive mutant Phm proteins still bind to a peptidylglycine substrate affinity resin. EPR and EXAFS studies of oxidized Phm indicate that the active site contains type 2 copper in a tetragonal environment; the copper is coordinated to two to three His and one to two additional O/N ligands, probably solvent, again supporting the structural homology of Phm and D beta M. Mutation of the Met residues common to Phm and D beta M to Ile identifies Met314 as critical for catalytic activity (Eipper, 1995).
Many neuropeptides and peptide hormones require amidation at the carboxyl terminus for activity. Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of these diverse physiological regulators. The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (Phm) with two coppers that cycle through cupric and cuprous oxidation states. The anomalous signal of the endogenous coppers was used to determine the structure of the catalytic core of oxidized rat Phm with and without bound peptide substrate. These structures strongly suggest that the Phm reaction proceeds via activation of substrate by a copper-bound oxygen species. The mechanistic and structural insight gained from the Phm structures can be directly extended to dopamine beta-monooxygenase (Prigge, 1997).
Peptidylglycine alpha-hydroxylating monooxygenase (Phm) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of Phm (Phmcc), refined to residues 42-356 of the Phm protein, is expressed at high levels in CHO (DG44) (dhfr-) cells. Phmcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified Phmcc under nonreducing conditions cleaves the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled Phmcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increases the Km of Phm for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increases the Km 8-fold and decreases the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in Phm, a two-domain model is proposed with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important (Kolhekar, 1997b).
Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (Phm; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the Phm reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. The Phm catalytic core (Phmcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the Phm catalytic cycle (Prigge, 1999).
Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the carboxyl-terminal amidation of bioactive peptides through a two-step reaction involving the monooxygenase and lyase domains. PAM undergoes endoproteolytic cleavage in neuroendocrine cells in the lyase domain. To determine which of the two possible paired basic sites is utilized, truncated PAM proteins ending at these sites were stably expressed in Chinese hamster ovary cells. While characterizing the truncation mutants, it became apparent that N-glycosylation occurs post-translationally at the single site localized near the carboxyl terminus of the lyase domain. The post-translational N-glycosylation of this site does not require the newly synthesized protein to remain tightly bound to membranes. Both malfolded, secretion incompetent proteins and fully active, secreted proteins are subject to post-translational N-glycosylation (Kolhekar, 1998).
Mechanisms underlying the specificity and efficiency of enzymes that modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. The process of peptide alpha-amidation was examined in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM) that contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides: this observation underscores the critical importance of regulation of peptide alpha-amidation (Spijker, 1999).
Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, it was unexpectedly observed that Charles River Sprague Dawley rats express forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats do not. Forms of PAM containing exon A are expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differs between rat breeds, and full-length PAM-1 is not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, do not vary with rat breed. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance (Ciccotosto, 2000).
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