Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: implications for PTHrP peptide production in lung cancer cells

The parathyroid hormone-related protein (PTHrP) precursor requires proteolytic processing to generate PTHrP-related peptide products that possess regulatory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically active peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multibasic, dibasic, and monobasic residues. These basic residues are predicted to serve as proteolytic cleavage sites for converting the PTHrP precursor into active peptide products. The coexpression of the prohormone processing enzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cells, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study, PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MALDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites to generate biologically active PTHrP-related peptides that correspond to the NH2-terminal domain (residues 1-37) that possesses PTH-like and growth regulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that modulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage sites. Overall, these results demonstrate the ability of PTP to cleave the PTHrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreactivity in PTHrP-containing lung cancer cells suggests PTP as a candidate processing enzyme for the PTHrP precursor.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles

Pro-opiomelanocortin (adrenocorticotropin/endorphin prohormone) is processed to yield active hormones by cleavages at paired basic amino acid residues. In this study, an enzyme that specifically cleaves at the paired basic residues of this prohormone has been purified from bovine pituitary intermediate lobe secretory vesicles, the intracellular processing site of proopiomelanocortin. This enzyme, named pro-opiomelanocortin converting enzyme, has been characterized as a glycoprotein of Mr approximately 70,000. It has an apparent isoelectric point between 3.5 and 4.0. The pH optimum of the pro-opiomelanocortin converting enzyme is between 4 and 5, but the enzyme is highly active at the intravesicular pH of 5.1-5.6. The enzyme specifically cleaved the Lys-Arg pairs of pro-opiomelanocortin to yield Mr = to 21,000-23,000 ACTH, beta-lipotropin, Mr 13,000 and 4,500 ACTH, beta-endorphin, and a Mr = 16,000 NH2-terminal glycopeptide, the products synthesized by the pituitary intermediate lobe in situ. NH2- and COOH-terminal analysis of the products indicated that the pro-opiomelanocortin converting enzyme cleaves the peptide bond either between the Lys and Arg or on the carboxyl side of the Arg at Lys-Arg pairs of pro-opiomelanocortin. The intracellular localization, pH optimum, and cleavage specificity of the enzyme suggest that it may function as a pro-opiomelanocortin processing enzyme in the pituitary intermediate lobe in vivo.     

The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B.

Purified cathepsin B from porcine parathyroid glands was allowed to act upon radioactive bovine parathormone and proparathormone at various ratios of enzyme to substrate and for different times. The reaction products were isolated by ion exchange chromatography and analyzed by gel electrophoresis, amino acid composition, sequence analysis, and bioassay. The enzyme cleaved parathormone between residues 36 and 37 yielding a major carboxyl and amino fragment and appeared to cleave proparathormone at the same locus. The amino fragments were degraded further by removal of small peptides (possibly, di- or tripeptides) from their COOH termini. In contrast there was little if any degradation of the carboxyl fragment (residues 37 to 84). Despite the ease with which the enzyme cleaved the arginyl bond in the synthetic substrate benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide, it did not remove the near homologous NH2-terminal hexapeptide extension of proparathormone (Lys-Ser-Val-Lys-Lys-Arg-R)--a reaction that would lead to the formation of parathormone from proparathormone. Purified liver cathepsin B cleaved the hormonal substrates in a fashion identical with that of the parathyroid enzyme.     

A Novel Type of Substrate Specificity of Rice BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase) with Mixed Endopeptidase and Carboxypeptidase

The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984)     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.     

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37 degrees C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.     

Enzymes that process somatostatin precursors. A novel endoprotease that cleaves before the arginine-lysine doublet is involved in somatostatin-28 convertase activity of rat brain cortex

The selective processing activity which generates both the NH2- and COOH-terminal fragments of the octacosapeptide somatostatin-28 (S-28) was investigated. Separation into two distinct proteolytic activities was achieved by ion-exchange chromatography. An endoprotease cleaving either the substrate Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr-NH2, i.e. [Ala17,Tyr20]S-28-(10-20)-NH2 (peptide I), or the octacosapeptide somatostatin-28, on the NH2 side of the Arg-Lys doublet was separated from an aminopeptidase B-like activity. Whereas the endoprotease cleaves a single peptide bond, between Glu12 and Arg13 of S-28, the aminopeptidase B-like enzyme removes both Arg13 and Lys14 stepwise from the NH2 terminus of the corresponding COOH-terminal fragment. This endoprotease activity peaks around pH 8.5, whereas the optimal aminopeptidase B-like activity is in the pH range 6.2-8.5. Combination of both enzymes resulted in the recovery of the overall S-28 convertase activity with an optimal pH at 7. In addition, this endoprotease appears to be very sensitive to divalent cations since it is strongly inhibited by chelating agents. The use of selectively modified undecapeptides derived from the reference substrate peptide I by a single modification of the amino acids Glu12, Arg13, and Lys14 at the cleavage locus showed that both basic residues are critically important, whereas Glu12 is not. It is proposed that S-28 processing involves a divalent cation-sensitive endoprotease that is sensitive to thiol reagents, which cleaves before the Arg-Lys doublet, which is not trypsin-like, and whose action is coupled to an aminopeptidase B-like enzyme.     

Characterization of an endoprotease from rat small intestinal mucosal secretory granules which generates somatostatin-28 from prosomatostatin by cleavage after a single arginine residue

We have extracted, characterized, and partially purified an enzyme from secretory granules from rat small intestinal mucosa which cleaves a synthetic prosomatostatin substrate on the carboxyl side of a single arginine residue. This substrate Leu-Gln-Arg-Ser-Ala-Asn-Ser-NH2 contains the monobasic site at which mammalian prosomatostatin is cleaved in vivo to generate somatostatin-28. This activity was released from the granules by osmotic shock followed by extraction with 500 mM KCl. The enzyme had a molecular weight of about 55,000, a pH optimum of about 7.5, and a Km for the synthetic substrate of 20 microM. It was partially inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, iodoacetate, soybean trypsin inhibitor, and EDTA. It was also very sensitive to aprotinin (complete inhibition at 25 micrograms/ml) but was not inhibited by bestatin, pepstatin, or p-chloromercuribenzoate. This endoprotease was unable to cleave three small trypsin and kallikrein substrates (N alpha-benzoyl-L-arginine ethyl ester, N alpha-benzoyl-DL-arginine p-nitroanilide, and N alpha-benzoyl-L-arginine 7-amido-4-methylcoumarin). It was unable to cleave either the Arg-Asp bond in CCK 12 or the Arg-Glu and Arg-Met bonds of synthetic peptides corresponding to sequences of anglerfish prosomatostatin II situated upstream from the somatostatin-28 domain. These observations together suggest that adjacent amino acids play a role in determining the conformational specificity of the monobasic cleavage. This soluble enzyme was also able to cleave three synthetic substrates containing dibasic residues (Arg-Lys or Lys-Arg) on the carboxyl side of the arginine, although it did so less rapidly than at the monobasic cleavage sites. When incubated with partially purified prosomatostatin from anglerfish pancreas, significant quantities of somatostatin-28 II were produced. All these cleavages were completely blocked by preincubation with aprotinin. Although further work is required to clarify the physiological role of this enzyme, it appears, in view of its catalytic properties, this endoprotease could be involved in the conversion of prosomatostatin to somatostatin-28 in intestine mucosal secretory cells.     

The amino acid sequence of Escherichia coli cyanase

The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.     

Treatment of an encephalitogenic peptide from guinea pig myelin basic protein with alpha-protease and thermolysin. Isolation of fragments and determination of cleavage sites.

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.     

Substrate specificity at the P1' site of Escherichia coli OmpT under denaturing conditions

Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg140-Arg141 and the modified substrates PRX substituted all 19 natural amino acids at the P1' site instead of Arg141. OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg140 except for the Arg140-Asp141, -Glu141, and -Pro141 pairs. In addition to Arg140 at the P1 site, similar results were obtained when Lys140 was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1' site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.     

Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora.

Neurospora NADP-specific glutamate dehydrogenase that was treated with iodoacetate, iodoacetamide, or N-ethylmaleimide to block the thiol groups was cleaved with cyanogen bromide. Of the expected 10 peptides, based on a methionine content of 9 residues, 8 were obtained in pure form and 2 were handled as a mixture. The fragments ranged in size from 9 to 109 residues. In addition, there were isolated 6 peptides, produced by anomalous cleavage at the carboxyl groups of tryptophan residues, and two by hydrolysis of an aspartyl-proline bond. Preliminary separation of these peptides was accomplished by gel filtration followed by either ion-exchange chromatography of the larger peptides or by paper chromatography and paper electrophoresis of the smaller fragments. Ordering of the CNBr fragments in sequence was based upon sequences of tryptic and chymotryptic peptides obtained in another laboratory. The complete sequence of the protein is presented. The amino acid sequences of the bovine and chicken liver glutamate dehydrogenases previously determined show considerable homology with the NADP-specific enzyme of Neurospora in the NH2-terminal half of the molecule; this includes the region of the specifically reactive lysine residue and the portion of the sequence that has been implicated in coenzyme binding. Particularly striking is the fact that most of the residues conserved among the three homologous proteins would be expected to be important for conformational, rather than catalytic, effects. This implies that the conformation of the Neurospora enzyme must be similar in parts of its structure to the vertebrate enzymes but undoubtedly differs in some regards.     

Structural basis of plasticity in protein tyrosine phosphatase 1B substrate recognition

Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.     

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.