Mode of action towards oligopeptides and proteins of hydrolase H, a high-molecular-weight aminoendopeptidase from rabbit skeletal muscle

The mode of action towards oligopeptides and proteins of hydrolase H purified from rabbit skeletal muscle was studied. The presence of protamine or alpha-N-benzoylarginine p-nitroanilide (an endopeptidase substrate) changed both the Km and V values of the enzyme towards Leu-beta-naphthylamide (an aminopeptidase substrate). This indicates that the binding site for an endopeptidase substrate is different from that for an aminopeptidase substrate. Hydrolase H as an aminopeptidase displayed broad specificity. The enzyme hydrolyzed various dipeptides readily except the dipeptides containing Pro or an amino acid with a hydrophobic beta-branched chain at the NH2 terminus. Pro and Val at the NH2 terminus of tripeptides were also difficult to release, whereas Ile and Val of tetrapeptides were easily released in contrast with those of dipeptides. The longer the peptide chain of Glyn (n = 2, 3, 4), the more susceptible was it to hydrolase H. Hydrolase H behaved as an endopeptidase only towards protamine among the proteins tested. The other proteins, casein, bovine serum albumin, myofibrils, troponin, hemoglobin, sarcoplasmic proteins, and myoglobin were probably attacked only by the aminopeptidase activity of the enzyme.     

Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.     

Isolation and functional properties of an arginine-selective endoprotease from rat intestinal mucosa. A putative prosomatostatin convertase.

The endoproteolytic activity previously detected in rat intestinal mucosal extracts (Beinfeld M., Bourdais, J., Kuks, P., Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 4460-4465), was purified to homogeneity as a 65-kDa molecular species. This putative proprotein-processing enzyme cleaves the peptide bond on the carboxyl side of a single arginine residue in hepta-[Leu62-Gln-Arg-Ser-Ala-Asn-Ser68] or trideca-[Asp56-Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg-Ser-Ala-Asn-+ ++Ser68] peptides, reproducing the prosomatostatin sequence around Arg64, the locus for endoproteolytic release of either somatostatin-28 or its NH2-terminal fragment, somatostatin-28-(1-12), from their common precursor. This enzyme exhibits a strict selectivity for arginyl residues, as demonstrated with related substrates, and did not cleave at lysyl residues. Moreover, only arginyl residues belonging to peptides of the prosomatostatin family were cleaved, since no hydrolysis of peptides from other prohormones was detected. In addition, the arginine residue situated at position -5 on the NH2-terminal side of Arg64 not only did not function as a cleavage locus, but had no effect on the overall cleavage kinetics of the prosomatostatin-(56-68) peptide substrate. This enzyme also cleaved, but with much less efficiency, the peptide bond on the carboxyl side of an arginine in peptides containing either an Arg-Lys or a Lys-Arg doublet corresponding to prohormone cleavage sites. This enzyme was insensitive to divalent cation chelators, was completely inhibited by aprotinin and leupeptin, and was somewhat inhibited by other serine-protease inhibitors. It is concluded that this endoprotease is a serine protease and could be involved in prohormone or proprotein post-translational processing at single arginine cleavage sites.     

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.     

A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.     

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.     

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.     

Structural and immunological evidence for the identity of prolyl aminopeptidase with leucyl aminopeptidase

Prolyl aminopeptidase (EC 3.4.11.5) has been assumed to be a unique enzyme catalyzing specifically the removal of unsubstituted NH2-terminal L-prolyl residues from various peptides and to be distinct from leucyl aminopeptidase (EC 3.4.11.1). In the present study, prolyl aminopeptidases were purified to apparent homogeneity from pig small intestine mucosa and human liver and their NH2-terminal amino acid sequences were determined together with that of pig kidney leucyl aminopeptidase. The NH2-terminal 24-residue sequence of pig intestinal prolyl aminopeptidase was shown to be identical with that of pig kidney leucyl aminopeptidase. The NH2-terminal sequence of human liver prolyl aminopeptidase was also shown to be very similar to that of pig kidney leucyl aminopeptidase. Further, pig intestinal prolyl aminopeptidase and pig kidney leucyl aminopeptidase were immunologically indistinguishable. These lines of evidence strongly suggest that prolyl aminopeptidase is identical with leucyl aminopeptidase.     

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.     

A Novel Aminopeptidase with Highest Preference for Lysine

Neuropeptides are formed from sedentary precursors to smaller, active peptides by processing enzymes cleaving at paired basic residues. The process generates peptide intermediates with additional Lys or Arg residues at their NH(2) and COOH termini; the N-terminal basic amino acids are later removed by specific aminopeptidases. We report here a novel lysine-specific aminopeptidase (KAP) of ubiquitous distribution. The enzyme was resolved from puromycin-sensitive aminopeptidase (PSA), aminopeptidase B (APB), and neuron-specific aminopeptidase (NAP). It was purified by FPLC after (NH(4))(2)SO(4) precipitation. The purified KAP had a K(m) of 333 microM with a V(max) of 0.7 nmol Lys ssNA/min/mg protein. N-terminal basic amino acids, Lys in particular, were its favorable substrates. KAP was inhibited by chelating agents and by serine protease inhibitors. It was highly sensitive to aminopeptidase inhibitor bestatin, but insensitive to puromycin and amastatin, showing that KAP is distinct from PSA, NAP, and aminopeptidase A (APA). The 62,000-Da enzyme had a pH optimum at 7.5 and NaCl was its strongest activator. However, metals could not restore KAP's activity after it was dialyzed against EGTA. Our data indicated that rat KAP did not resemble any aminopeptidases as well as the microbial lysine aminopeptidases.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

Specificity of the surface aminopeptidase in Moniliformis moniliformis (Acanthocephala)

The hydrolysis of various oligopeptides in solution by intact Moniliformis moniliformis was examined using paper chromatographic analysis of the incubation medium. In the presence of transport inhibitors, the respective peptide sub-units and/or amino acid residues accumulated in the bathing medium. Only peptides with serine, methionine, leucine or alanine at the NH2-terminal end of the peptide were hydrolysed. There was no hydrolysis when these amino acids were located internally or at the COOH-terminus indicating genuine aminopeptidase activity of the class, alpha-aminoacylpeptide hydrolase. Hydrolysis was negligible when the NH2-terminus was arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, phenylalanine, proline, tryptophan, tyrosine, or valine. In separate experiments, mediated uptake of 0.1 mM 3H-leucine by the worms in 2 min was inhibited 100% by 5 mM unlabelled leucine or tri-serine, but only partially inhibited by 5 mM Ser-Gly (66%), 10 mM Ser-Gly (74%), 5 mM Leu-Leu (69%), 10 mM Leu-Leu (70%), 5 mM Leu-Gly (58%) or 5 mM Met-Met (69%). Because the inhibitions produced by 5 mM Leu-Leu plus 5 mM Met-Met (79%) or 5 mM Leu-Leu plus 5 mM Ser-Gly (76%) were not additive, a single enzyme is indicated. The name serine aminopeptidase is proposed because of its preference for serine.     

Porcine spleen cathepsin B is an exopeptidase

The major cathepsin B isozyme CB-I purified from porcine spleens was studied for its specificity against various peptide and denatured protein substrates. The enzyme degraded all the peptide substrates by an exopeptidase activity. The substrates were degraded mainly by a dipeptidyl carboxypeptidase activity of the enzyme except for angiotensin I, from which a COOH-terminal leucine residue was released. The enzyme failed to hydrolyze peptides having a proline or cysteic acid in the COOH-terminal, penultimate, and antepenultimate positions. Reduced and carboxymethylated soybean trypsin inhibitor was degraded by the same dipeptidyl carboxypeptidase action of cathepsin B. No significant endopeptidase activity was observed. These results do not support the general assumption that cathepsin B has both endo- and exopeptidase activities, neither do these observations support the postulation that cathepsin B might be involved in the in vivo proteolytic processing of protein precursors. We propose that the biological role of this enzyme is mainly the degradation of tissue proteins in lysosomes.     

Acylpeptide hydrolase activity from erythrocytes

Acylpeptide hydrolase, which cleaves the NH2-terminal acetylated or formylated amino acid from a blocked peptide, has been purified to apparent homogeneity from human erythrocytes. The enzyme catalyzes the hydrolysis of a diverse number of peptides and displays different pH optima for certain substrates in doing so. Zinc inhibits to the same extent the hydrolysis of both the most efficient and the least efficient substrates. This enzyme may play a pivotal role in the processing of polypeptide chains during biosynthesis.     

Purification and characterization of proline-beta-naphthylamidase, a novel enzyme from pig intestinal mucosa

An enzyme hydrolyzing proline-beta-naphthylamide was purified to apparent homogeneity from porcine intestinal mucosa. The purified enzyme appears to consist of three identical subunit polypeptides with a molecular weight of about 58,000 each, associated noncovalently. The enzyme is a glycoprotein, and the subunit polypeptide contains 3 residues each of mannose and N-acetylglucosamine. A wide variety of peptidase substrates were tested for the enzyme, and the results showed that it hydrolyzes only aminopeptidase substrates, such as proline-beta-naphthylamide, glycine-beta-naphthylamide, leucine-beta-naphthylamide, and alanine-beta-naphthylamide. Among these substrates, proline-beta-naphthylamide is most efficiently hydrolyzed as judged by the kcat/Km value. The optimum pH for this substrate is around 9. The enzyme also hydrolyzes efficiently the ester substrates of these amino acids. No hydrolytic activity was observed for the peptide and protein substrates tested. The proline-beta-naphthylamidase activity was drastically inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, indicating that the enzyme is a serine hydrolase, whereas it was slightly inhibited by aminopeptidase inhibitors, such as amastatin, bestatin, and puromycin. No significant homology was found for the NH2-terminal sequence of 27 amino acid residues with any known protein sequences. From these results we conclude that the enzyme is a protein which has not been described before.     

Activation of human plasminogen by equimolar levels of streptokinase.

Native Glu-human plasminogen (Mr approximately 92,000 with NH2-terminal glutamic acid) is able to combine directly with streptokinase in an equivalent molar ratio, to yield a stoichiometric complex. The plasminogen moiety in the complex then undergoes streptokinase-induced conformational changes. As a result of such, an active center develops in the plasminogen moiety of the complex. This proteolytically active complex then activates plasminogen in the complex to plasmin and at least two peptide bonds are cleaved in the process. The data presented in this paper reveal that initially an internal peptide bond of plasminogen (in the complex) is cleaved to yield a two-chain, disulfide-linked plasmin molecule. The heavy chain (Mr approximately 67,000 with NH2-terminal glutamic acid) of this plasmin molecule has an identical NH2-terminal amico acid as the native plasminogen. The light chain (Mr approximately 25,000 with NH2-terminal valine) of plasmin is known to be derived from the COOH-terminal portion of the parent plasminogen molecule. A second peptide is then cleaved from the NH2-terminal end of the heavy chain of plasmin producing a proteolytically modified heavy chain (Mr =60.000 with NH2-terminal lysine). This cleavage of the NH2-terminal peptide from the heavy chain of plasmin is shown to be mediated by the dissociated free plasmin present in the activation mixture. Plasmin in the streptokinase-plasmin complex is unable to cleave this NH2-terminal peptide. This same NH2-terminal peptide can also be cleaved from native Glu-plasminogen or from the Glu-plasminogen-streptokinase complex by free plasmin and not by a complex of streptokinase-plasmin. From these studies we conclude (a) in the streptokinase-plasminogen complex, the NH2-terminal peptide need not be released prior to the cleavage of the essential Arg-Val peptide bond which leads to the formation of a two chain plasmin molecule and (b) that this peptide is cleaved from the native plasminogen or from the heavy chain of the initially formed plasmin in the streptokinase complex by free plasmin and not by the plasmin associated with streptokinase. In agreement with this, plasmin associated with streptokinase was unable to cleave the NH2-terminal peptide from the isolated native heavy chain possessing glutamic acid as the NH2-terminal amino acid; whereas free plasmin readily cleaved this peptide from the same isolated Glu-heavy chain.     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase.

Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the 'entrance' of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2-9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.     

Structure of the agonist-binding site of the nicotinic acetylcholine receptor. [3H]acetylcholine mustard identifies residues in the cation-binding subsite.

To characterize the structure of the agonist-binding site of the Torpedo nicotinic acetylcholine receptor (AChR), we have used [3H]acetylcholine mustard [( 3H]AChM), a reactive analog of acetylcholine, to identify residues contributing to the cation-binding subsite. Reaction of [3H]AChM, in its aziridinium form, with AChR-rich membrane suspensions, resulted initially in reversible, high affinity binding (K approximately 0.3 microM) followed by slow alkylation of the acetylcholine-binding site. Incorporation of label into AChR alpha-subunit was inhibited by agonists and competitive antagonists, but not by noncompetitive antagonists, and reaction with 3 microM [3H]AChM for 2 h resulted in specific alkylation of 0.6% of alpha-subunits. Within the alpha-subunit, greater than 90% of specific incorporation was contained within an 18-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Val-46 and containing N-linked carbohydrate. To identify sites of specific alkylation, [3H]AChM-labeled alpha-subunit was digested with trypsin, and the digests were fractionated by reverse phase high pressure liquid chromatography. Specifically labeled material was recovered within a single peak containing a peptide extending from Leu-80 to Lys-107. NH2-terminal amino acid sequencing revealed specific release of 3H in cycle 14 corresponding to alpha-subunit Tyr-93. Identification of Tyr-93 as the site of alkylation was confirmed by radiosequence analysis utilizing o-phthalaldehyde to establish that the released 3H originated from a peptide containing prolines at residues 2 and 9. Because [3H]AChM contains as its reactive group a positively charged quaternary aziridinium, alpha-subunit Tyr-93 is identified as contributing to the cation-binding domain of the AChR agonist-binding site. The selective reaction of [3H]AChM with tyrosyl rather than acidic side chains indicates the importance of aromatic interactions for the binding of the quaternary ammonium group, and the lack of reaction with the tyrosyl or acidic side chains within alpha 190-200 emphasizes the selective orientation of acetylcholine within its binding site.