Ubiquitin and ubiquitin-derived peptides as substrates for peptidylglycine alpha-amidating monooxygenase

Ubiquitin (Ub) and the ubiquitin-like proteins (UBLs) mediate an array of cellular functions. These proteins contain a C-terminal glycine residue that is key to their function. Oxidative conversion of C-terminal glycine-extended prohormones to the corresponding alpha-amidated peptide is one step in the biosynthesis of bioactive peptide hormones. The enzyme catalyzing this reaction is peptidylglycine alpha-amidating monooxygenase (PAM). We report herein that Ub is a PAM substrate with a (V/K)(amidation) that is similar to other known peptide substrates. This work is significant because PAM and the UBLs co-localize to the hypothalamus and the adrenal medulla and are both over-expressed in glioblastomas.     

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.     

Peptide C-terminal alpha-amidating enzyme purified to homogeneity from Xenopus laevis skin

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. In this study, we have developed a simple and sensitive assay for monitoring alpha-amidating activity by using radioiodinated Ac-Tyr-Phe-Gly as a substrate. By utilizing this assay, an alpha-amidating enzyme was first purified to homogeneity from Xenopus laevis skin. The purified enzyme has a single polypeptide chain with an apparent molecular weight of 39,000 and its N-terminal sequence was determined as Ser-Leu-Ser-. The enzyme converts several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides. The enzyme activity, with an optimal pH 6-7, was dependent on the copper ion and ascorbate. In the presence of 0.25 mM ascorbate, the enzyme exhibited a Km of 0.35 microM and a Vmax of 1.9 nmol/microgram/h for Ac-Tyr-Phe-Gly.     

Purification and characterization of a peptide C-terminal alpha-amidating enzyme from porcine atrium

In many peptide hormones and neuropeptides, the carboxy-terminal alpha-amide structure is essential in eliciting biological activity. Here we report the purification and characterization of an alpha-amidating enzyme from porcine atrium, in which a high concentration of alpha-amidating activity was detected. The enzyme was purified to homogeneity from the membrane fraction of porcine atria by five steps of chromatography, including an affinity chromatography using a Sepharose column coupled with a substrate, Tyr-Phe-Gly. The purified enzyme was found to be composed of a single polypeptide chain with an apparent molecular weight of 92,000. This enzyme converted several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides.     

N-acylglycine amidation: implications for the biosynthesis of fatty acid primary amides

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the O2-dependent conversion of C-terminal glycine-extended prohormones to the active, C-terminal alpha-amidated peptide and glyoxylate. We show that alpha-AE will also catalyze the oxidative cleavage of N-acylglycines, from N-formylglycine to N-arachidonoylglycine. N-Formylglycine is the smallest amide substrate yet reported for alpha-AE. The (V/K)app for N-acylglycine amidation varies approximately 1000-fold, with the (V/K)app increasing as the acyl chain length increases. This effect is largely an effect on the KM,app; the KM,app for N-formylglycine is 23 +/- 0.88 mM, while the KM,app for N-lauroylglycine and longer chain N-acylglycines is in the range of 60-90 microM. For the amidation of N-acetylglycine, N-(tert-butoxycarbonyl)glycine, N-hexanoylglycine, and N-oleoylglycine, the rate of O2 consumption is faster than the rate of glyoxylate production. These results indicate that there must be the initial formation of an oxidized intermediate from the N-acylglycine before glyoxylate is produced. The intermediate is shown to be N-acyl-alpha-hydroxyglycine by two-dimensional 1H-13C heteronuclear multiple quantum coherence (HMQC) NMR.     

Peptide substrate specificity of the alpha-amidating enzyme isolated from rat medullary thyroid CA-77 cells

The kinetic parameters were obtained for enzymatic alpha-amidation of peptides of the form N-dansyl-(Gly)4-X-Gly-OH, in which the amino acid at position X was substituted with each of the 20 natural amino acids. The enzyme used in these studies was a highly enriched preparation of alpha-amidating enzyme secreted by a clonal (CA-77) cell line which actively expresses mature alpha-amidated peptides. A 130-fold and 11-fold variation respectively in apparent Km and Vmax values was observed. The effect of the amino acid side chain at position X in stabilization of the enzyme-substrate complex decreased through the series X = planar aromatic or sulfur containing greater than neutral aliphatic greater than polar and basic greater than cyclic aliphatic or acidic.     

Human C5a-related synthetic peptides as neutrophil chemotactic factors.

The pentapeptide L-methionyl-L-glutaminyl-L-leucyl-glycyl-L-arginine, which mimics the C-terminal sequence of human C5a anaphylatoxin, and two additional N-formylmethionyl derivatives of this peptide have been assessed for their ability to simulate C5a-related biological activities. Only the N-formylated peptides display chemotactic activity or induce lysosomal enzyme release when assayed with human neutrophils. Additional studies indicate that the active peptides, although designed after the C-terminal structure of the human C5a molecule, were apparently active because of their interaction with the N-formylmethionyl peptide receptor rather than the C5a receptor on neutrophils.     

The reaction product of peptidylglycine alpha-amidating enzyme is a hydroxyl derivative at alpha-carbon of the carboxyl-terminal glycine

The peptidylglycine alpha-amidating enzyme catalyzes a reaction that transforms a carboxyl-terminal glycine-extended precursor into a carboxyl-terminal alpha-amidated peptide. We purified an alpha-amidating enzyme from equine serum by simplified steps including substrate affinity chromatography. With the purified enzyme, we detected an intermediate of the alpha-amidating reaction by high performance liquid chromatography analysis. The production of the intermediate required copper, oxygen, and ascorbate and increased linearly with incubation time. The structure of the intermediate was determined to be a hydroxyl derivative at the carboxyl-terminal glycine by fast atom bombardment mass spectrometry and by proton NMR. The intermediate was readily converted into an alpha-amidated product in alkaline conditions in a nonenzymic fashion. The nonenzymic conversion required no cofactor but was extremely accelerated by the addition of copper ion or at higher temperature. Our data suggest that the direct product of the alpha-amidating reaction is not an alpha-amidated peptide but a hydroxyl derivative at the alpha-carbon of the carboxyl-terminal glycine.     

Glutathione,S-substituted glutathiones,and leukotriene C4 as substrates for peptidylglycine alpha-amidating monooxygenase

The C-terminal alpha-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine alpha-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C(4) results in the consumption of O(2) and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal alpha-hydroxyglycine-extended peptide. Amidated glutathione (gammaGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V/K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest (V/K)(app) values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V/K)(app) of the substrates described in this report.     

Prohormone-substrate peptide sequence recognition by peptidylglycine α-amidating monooxygenase and its reflection in increased glycolate inhibitor potency

The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied. The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme extends more than four and five amino acid residues, respectively, from their C-termini. This substrate sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the glycolate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of prooxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity. However, this changes the binding mode from one that depends on peptide sequence recognition to another primarily determined by the phenylalanine moiety, for both the substrates and analogous glycolate inhibitors.     

Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease.

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a beta-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 A N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba67,95]HIVPR and [Lys7,Ile33,Aba67,95]HIVPR used in this work were shown to have very similar crystal structures.     

Cloning of cDNA encoding a new peptide C-terminal alpha-amidating enzyme having a putative membrane-spanning domain from Xenopus laevis skin

A cDNA clone encoding a precursor of a peptide C-terminal alpha-amidating enzyme (AE-I) from Xenopus laevis skin was recently isolated and sequenced in our laboratory. In this study, by using the restriction fragment of this clone as a hybridization probe, we have identified the cDNA encoding another new peptide C-terminal alpha-amidating enzyme (tentatively named AE-II) distinct from AE-I. The cDNA encodes a polypeptide of 875 amino acid residues, which contains a region extensively homologous to AE-I precursor at N-terminus. The encoded protein characteristically has a putative membrane-spanning domain near C-terminus. Our results indicate that C-terminal alpha-amide formation of peptides in Xenopus skin is regulated by at least two distinct alpha-amidating enzymes.     

Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.     

New actinomycin from Actinomyces sp. No. 2

A new actinomycin was isolated from a mixture of actinomycins formed by Actinomyces sp. No. 2, an organism producing auranthin, an actinomycetous antibiotic. The peptide chains of the new actinomycin contain such amino acids as threonine, valine, proline and sarcosine in a ratio of 2 : 4 : 2 : 2. N-Methyl-valine characteristic of all actinomycins is replaced in position 5 of both pentapeptide chains of the new actinomycin by valine. The new actinomycin is actinomycin D undermethylated in position 5 by valine. When the growing culture of Actinomyces olivobrunneus producing actinomycin D was exposed to sulfadimesine, an inhibitor of biological methylation, production of actinomycin D0 (sarcosine replaced by glycine in one of the pentapeptide chains) markedly increased, which indicated impairment of the glycine residue methylation. Still, no impairment of the valine residue methylation in position 5 of the pentapeptide chains was observed an no actinomycin with N-methyl-valine replaced by valine was formed.     

Further characterization of the peptidyl alpha-amidating enzyme in rat anterior pituitary secretory granules

In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha-melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides.     

The extended active site of guinea pig liver transglutaminase.

The catalytic activities of guinea pig liver transglutaminase toward glutamine-containing peptide derivatives of three series have been studied. These series include: (a) formylheptapeptides of the basic structure, HCO-GLY3-L-Gln-Gly3. A single L-leucine residue was systematically substituted for glycine at a different position in each peptide; (b) formyltripeptides of the basic structure, HCO-Gly-L-Gln-Gly. L-Leucine was substituted for glycine in each position and in both positions; (c) various N-acyl derivatives of the dipeptide, L-Gln-Gly. Comparison of the values of the kinetic constants for methylamine incorporation and for hydroxylamine incorporation with the peptide derivatives shows that the length of the peptide chain has a pronounced influence on catalysis, as does the position of the leucine residue in the longer chain peptide derivatives. The kcat/Km(app) values for each substrate calculated from data for methylamine incorporation and from those for hydroxylamine incorporation were found to be in good agreement. However, both the observed maximum velocity and the apparent Michaelis constant for each peptide derivative were significantly larger for hydroxylamine incorporation than for methylamine incorporation. Interpretation of these findings as evidence for a normal catalytic mechanism for each amine incorporation reaction and for the limiting nature of deacylation to methylamine is discussed. Two observations caution against such an interpretation. These are the significantly higher inhibitor constants found fo formylhexaglycine and for several other competitive inhibitors in the hydroxylamine incorporation reaction, and earlier findings of higher turnover values with hyroxylamine in cases were acylation appears to be limiting for methylamine incorporation. Methods of preparation, supporting analytical data and properties of the peptide intermediates, the peptides, and their derivatives used in this study are presented in the miniprint supplement immediately following this paper.     

Peptide thioesters and 4-nitroanilides as substrates for porcine pancreatic kallikrein

A series of 14 4-nitroanilide substrates and 17 thioester substrates have been used to measure kinetic constants with porcine pancreatic kallikrein. All of the substrates have a P1 arginine residue. The 4-nitroanilide substrates consist of seven P2-glycine and seven P2-phenylalanine tripeptides. As expected from previous results, the phenylalanine series substrates were generally 100-fold 'better' than those in the glycine series. The S3 subsite was found to 'prefer' lysine or phenylalanine, whereas glutamic acid in this position was distinctly unfavourable. The thioester substrates consisted of various thioester derivatives of arginine as well as 12 dipeptides. These substrates exhibited kcat./Km values generally 1000 times higher than the P2-phenylalanine 4-nitroanilides. With the thioesters, a P2 phenylalanine or tryptophan residue yielded the best substrates, but some of the simple derivatives of arginine were nearly as good. A comparison of the kinetic constants of the thioester substrates between the porcine enzyme and human plasma kallikrein provides further evidence that these enzymes have a similar preference for bulky P2 residues, but otherwise are quite different enzymes. The thioester substrates are nearly as reactive as oxygen ester substrates such as acetylphenylalanylarginine methyl ester for the porcine enzyme [Levison & Tomalin (1982) Biochem. J. 203, 299-302; Fiedler (1983) Adv. Exp. Med. Biol. 156A, 263-274], and owing to the greater ease in assaying with the thioesters, they should find use in routine assays for the glandular kallikreins.     

Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence similarity between dopamine beta-hydroxylase and peptide alpha-amidating enzyme: evidence for a conserved catalytic domain

A comparison of human dopamine beta-hydroxylase (EC 1.14.17.1) with bovine peptide C-terminal alpha-amidating enzyme (EC 1.14.17.3), revealed a 28% identity extending throughout a common catalytic domain of approximately 270 residues. The shared biochemical properties of these two enzymes from neurosecretory granules suggests that the sequence similarity reflects a genuine homology and provides a structural basis for a new family of copper type II, ascorbate-dependent monooxygenases.     

Structure and activity of angiotensin I converting enzyme inhibitory peptides derived from Alaskan pollack skin

Angiotensin I that converts the enzyme (ACE) inhibitory peptide, Gly-Pro-Leu, previously purified and identified from the Alaskan pollack skin gelatin hydrolysate, were synthesized. In addition, the peptides Gly-Leu-Pro, Leu- Gly-Pro, Leu-Pro-Gly, Pro-Gly-Leu, Pro-Leu-Gly, Gly- Pro, and Pro-Leu, which consisted of glycine, proline, and leucine, were synthesized by the solid-phase method. The IC50 values of each tripeptide. namely Leu-Gly-Pro, Gly- Leu-Pro, Gly-Pro-Leu, Pro-Leu-Gly, Leu-Pro-Gly, and Pro-Gly-Leu. were 0.72, 1.62, 2.65, 4.74, 5.73, and 13.93 microM, respectively. The ACE inhibitory activity of these tripeptides was higher than that of dipeptides, such as Gly- Pro and Pro-Leu with IC50 values of 252.6 and 337.3 microM, respectively. Among the tripeptides, Leu-Gly-Pro and Gly- Leu-Pro had higher inhibitory activity than Gly-Pro-Leu that was isolated from the Alaskan pollack skin gelatin hydrolysate. Among the different types of tripeptides that were examined, the highest ACE inhibitory activity was observed for Leu-Gly-Pro. It had the leucine residue at the N-terminal and proline residue at the C-terminal.