Aminopeptidase N from Streptococcus salivarius subsp. thermophilus NCDO 573: purification and properties

A 96 kDa aminopeptidase was purified from Streptococcus salivarius subsp. thermophilus NCDO 573. The enzyme had similar properties to aminopeptidases isolated from lactococci and lactobacilli and showed a high degree of N -terminal amino acid sequence homology to aminopeptidase N from Lactococcus lactis subsp. cremoris. It catalysed the hydrolysis of a range of aminoacyl 4-nitroanilides and 7-amido-4-methylcoumarin derivatives, dipeptides, tripeptides and oligopeptides. In common with aminopeptidases from other lactic acid bacteria, the enzyme from Strep. salivarius subsp. thermophilus showed highest activity with lysyl derivatives but was also very active with arginyl and leucyl derivatives. Relative activity with alanyl, phenylalanyl, tyrosyl, seryl and valyl derivatives was considerably lower and with glycyl, glutamyl and prolyl derivatives almost negligible. The aminopeptidase also catalysed the hydrolysis of dipeptides and tripeptides but mostly at rates much less than that with L-lysyl-4-nitroanilide and oligopeptides. The enzyme catalysed the successive hydrolysis of various amino acid residues from the N -terminus of several oligopeptides but it was unable to cleave peptide bonds on the N -terminal side of a proline residue.     

Enkephalin-degrading aminopeptidase in the longitudinal muscle layer of guinea pig small intestine: its properties and action on neuropeptides.

A membrane-bound enkephalin-degrading aminopeptidase was purified from the longitudinal muscle layer of the guinea pig small intestine by four steps of column chromatography using L-tyrosine beta-naphthylamide. The molecular weight of the enzyme was estimated to be 105,000 by gel filtration. The maximum activity was observed between pH 6.5 and 7.0. The Km value for leucine-enkephalin was 137 microM. The aminopeptidase activity toward aminoacyl beta-naphthylamide substrates was restricted to basic, neutral, and aromatic aminoacyl derivatives. No action was detected on acidic amino acid and proline derivatives. The enzyme was potently inhibited by the aminopeptidase inhibitors actinonin, amastatin, and bestatin, and bioactive peptides such as angiotensin III, substance P, and Met-Lys-bradykinin. The enzyme activity was also inhibited by the antibody against the purified serum enkephalin-degrading aminopeptidase of guinea pig at concentrations similar to those at which activity was observed toward serum enkephalin-degrading aminopeptidase and renal aminopeptidase M. The enzyme rapidly hydrolyzed Leu-enkephalin and Met-enkephalin with the sequential removal of the N-terminal amino acid residues. The enzyme also hydrolyzed two enkephalin derivatives, angiotensin III and neurokinin A. However, neurotensin, substance P, and bradykinin were not cleaved. These properties indicated that the membrane-bound enkephalin-degrading aminopeptidase in the longitudinal muscle layer of the small intestine is similar to the serum enkephalin-degrading aminopeptidase and resembles aminopeptidase M. It is therefore suggested to play an important role in the metabolism of some bioactive peptides including enkephalin in peripheral nervous systems in vivo.     

Characterization of the biochemical properties of two methionine aminopeptidases of Cryptosporidium parvum

We identified two methionine aminopeptidases of Cryptosporidium parvum (CpMetAP1 and CpMetAP2) and characterized the biochemical properties of the recombinant enzymes. CpMetAP1 and CpMetAP2 belong to the type I and type II MetAP subfamilies, respectively. Both CpMetAPs have typical amino acid residues essential for metal binding and substrate binding sites, which are conserved in the MetAP family. Bacterially expressed recombinant CpMetAP1 and CpMetAP2 showed similar biochemical properties including a broad optimal pH range (pH 7.5-8.5) with maximum activity at pH 8.0. The two enzymes were stable under neutral and alkaline pHs but were relatively unstable under acidic conditions. The activities of CpMetAP1 and CpMetAP2 increased highly in the presence of Mn(2+) and Co(2+). CpMetAP1 and CpMetAP2 were effectively inhibited by the metal chelators, EDTA and 1,10-phenanthroline, and were partially inhibited by the aminopeptidase inhibitors, amastatin and bestatin. Fumagillin also showed an inhibitory effect on both CpMetAPs.     

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.     

Homology modeling and site-directed mutagenesis of pyroglutamyl peptidase II. Insights into omega-versus aminopeptidase specificity in the M1 family

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound omegapeptidase, removes N-terminal pyroglutamyl from thyrotropin-releasing hormone (相似文献   

Insulin-regulated aminopeptidase: analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine beta-naphthylamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.     

Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.     

Comparison of aminopeptidase inhibition by amino acids in human and porcine skeletal muscle tissues in vitro

We compared the inhibitory action of individual amino acids in vitro on the activities of alanyl-, arginyl-, leucyl- and pyroglutamyl aminopeptidases purified from human and porcine skeletal muscle tissues. The range of susceptibility to inhibition by individual amino acids (<25 mM) for different aminopeptidase types broadly paralleled that for the respective substrate specificities (in terms of relative rates of hydrolysis of amino acyl-AMC derivatives) for these enzymes. Thus, alanyl aminopeptidase (which hydrolyses a broad range of aminoacyl-AMC substrates) was inhibited by a correspondingly broad range of amino acids (although the respective ranking order of amino acids was not identical in each case), whereas pyroglutamyl aminopeptidase (which hydrolyses only pyroglutamyl AMC as substrate) was inhibited by pyroglutamic acid only. The mode of inhibition (competitive/non-competitive) varied for different enzyme types, both within and between each species. For enzymes purified from human muscle, alanyl, arginyl and leucyl aminopeptidases were inhibited by amino acids via the non-competitive mode (pyroglutamyl aminopeptidase via the competitive mode), whereas corresponding enzymes purified from porcine muscle were inhibited via the competitive mode. The data obtained indicate that the same aminopeptidase types are present in human and porcine skeletal muscle tissues, with corresponding enzymes having broadly similar assay characteristics and susceptibilities to inhibition by amino acids (although the mode of inhibition for corresponding enzymes may differ in each species). Such data obtained in vitro may prove of value in devising experimental strategies to manipulate protein turnover/muscle deposition in vivo, via inhibition of aminopeptidase action after administration of an appropriate admixture of amino acids.     

Inhibitors of a Rat Brain Enkephalin Aminopeptidase

Abstract: Eight protease inhibitors of microbiological origin were examined as potential inhibitors of a homogeneous rat brain enkephalin aminopeptidase. Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]- l -leucine and analogs of bestatin having basic, acidic, and other neutral amino acids substituted for the Leu residue exhibited inhibition constants ranging from 3.3 ± 10⁻⁵ to 8.3 ± 10⁻⁸ m . The best inhibitor had a positively charged amino acid (Lys) substituted for Leu. A series of phenylalanyl dipeptides were examined as substrates with the aminopeptidase. The amino acid residue on the carboxyl side of the peptide bond undergoing cleavage was varied systematically in the dipeptides to include neutral, acidic, and basic residues. Again, a positively charged amino acid (Arg) adjacent to the bond undergoing scission was kinetically preferred. These results may be used to design highly specific inhibitors of the enkephalin aminopeptidase.     

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.     

Aminopeptidase Fingerprints, an Integrated Approach for Identification of Good Substrates and Optimal Inhibitors

Aminopeptidases process the N-terminal amino acids of target substrates by sequential cleavage of one residue at a time. They are found in all cell compartments of prokaryotes and eukaryotes, being implicated in the major proteolytic events of cell survival, defense, growth, and development. We present a new approach for the fast and reliable evaluation of the substrate specificity of individual aminopeptidases. Using solid phase chemistry with the 7-amino-4-carbamoylmethylcoumarin fluorophore, we have synthesized a library of 61 individual natural and unnatural amino acids substrates, chosen to cover a broad spectrum of the possible interactions in the S1 pocket of this type of protease. As proof of concept, we determined the substrate specificity of human, pig, and rat orthologs of aminopeptidase N (CD13), a highly conserved cell surface protease that inactivates enkephalins and other bioactive peptides. Our data reveal a large and hydrophobic character for the S1 pocket of aminopeptidase N that is conserved with aminopeptidase Ns. Our approach, which can be applied in principle to all aminopeptidases, yields useful information for the design of specific inhibitors, and more importantly, reveals a relationship between the kinetics of substrate hydrolysis and the kinetics of enzyme inhibition.     

Leukotriene A4 hydrolase, a bifunctional enzyme. Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297.

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu-297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His-296, His-300 and Glu-319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site-directed mutagenesis experiments were carried out. Glu-297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu-297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.     

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.     

Molecular evolution and zinc ion binding motif of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase belongs to the aminopeptidase N family. In order to investigate the molecular evolution and physiological significance of LTA4 hydrolase, the enzymes belonging to the family were aligned and a phylogenetic tree was constructed. From the alignment, it was found that three residues involved in zinc binding and one residue of the active sites of aminopeptidases N were conserved in LTA4 hydrolase. In agreement with the observation, LTA4 hydrolase is a zinc protein as determined by atomic absorption spectroscopy.     

Leukotriene A4 hydrolase is a zinc-containing aminopeptidase

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.     

Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum

During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.     

Chloride-insensitive, glycine-phenylalanine-naphthylamide hydrolysis at neutral pH in human skin fibroblasts

Crude lysosomal preparations from a cultured human skin fibroblast line were found to contain significant levels of a neutral pH hydrolase activity towards glycine--phenylalanine--beta-naphthylamide (NA), a substrate normally used for the assay of lysosomal dipeptidyl aminopeptidase I. However, the activity was chloride ion insensitive, nonlatent, and inhibitable by cationic detergents and amino acids. Assays of substrate selectivity, relative substrate affinity, pH and anion and cation sensitivity indicated the activity to be distinct from dipeptidyl aminopeptidases I (chloride-dependent hydrolysis of Pro-Phe-, Gly-Phe-, Gly-Arg-, and Pro-Arg-NA's at acid pH), II (Lys-Ala-NA hydrolysis), III (Arg-Arg-NA hydrolysis), and IV (Gly-Pro-NA hydrolysis). The lysosomal preparations also contained significant activity towards several amino acid--naphthylamides, notably Arg-NA. Only dipepidyl aminopeptidase I activity showed sensitivity to chloride anions, both dipeptidyl aminopeptidases I and II showed substantial latency, and none of the activities displayed a significant metal cation dependent.     

Effect of alkylation of tryptophan residues on the enzymatic and pharmacological properties of snake venom phospholipase A2

Snake venom phospholipases A2 show a remarkable degree of amino acid sequence homology yet differ markedly in enzymatic and pharmacological activities. The basic phospholipase A2 from Naja nigricollis venom has much greater lethal potency, cardiotoxicity, hemolytic and anticoagulant activity than the acidic or neutral enzymes from Naja naja atra or Hemachatus haemachatus venoms, respectively, even though it has lower enzymatic activity than the latter two enzymes. Previous studies in which we selectively modified lysine and free carboxyl groups suggested that the pharmacological and enzymatic active sites are not identical. Tryptophan residues have been suggested as being involved in substrate binding although some phospholipases have no tryptophan. We investigated the effect of alkylating the tryptophans in N. nigricollis, N. n. atra, and H. haemachatus phospholipases A2 with 2-hydroxy-5-nitrobenzyl bromide. Chemical modification caused decreases in enzymatic activity, although the extent of inactivation varied with the enzyme and with the substrate (lecithin micelles, egg yolk, heart homogenates). The specificity of the enzymes for individual phospholipid substrates was not affected. Alkylation of the tryptophans also caused decreases in lethal, hemolytic, anticoagulant, and cardiotoxic potencies, which were similar to the extents of decrease in enzymatic activity. Our results suggest that tryptophans are not specifically associated with either the enzymatic or the pharmacological active site nor are essential for either activity.     

Localization and characterization of soluble and plasma membrane aminopeptidase activities in Arabidopsis seedlings

We previously demonstrated that N,1-naphthylphthalamic acid is hydrolyzed at the root-hypocotyl transition zone and other regions of Arabidopsis thaliana seedlings, and that this reaction, like NPA-induced growth inhibition, is strongly promoted by blue light. In addition, NPA amidase activity was detected in plasma membrane-enriched fractions obtained from Arabidopsis seedlings. To further investigate this phenomenon, we tested the hypothesis that the arylamidase(s) responsible for NPA hydrolysis may also have aminopeptidase activity. The responses of Arabidopsis seedlings to various aminopeptidase substrates were tested. The hydrolysis of Tyr-, Trp-, Pro- and Gly-Pro-β-naphthylamide aminopeptidase substrates was shown to be histochemically localized at the root-hypocotyl transition zone and other regions where NPA hydrolysis also occurs. Blue light stimulated the in vivo activity of Tyr- and Pro-aminopeptidase activities, and far-red light stimulated the activity of the Trp-aminopeptidase. These same substrates also induced NPA-like growth inhibitory effects. In parallel experiments, aminopeptidase activities were detected in the supernatant and plasma membrane fractions of seedling extracts. The soluble AP activities resemble previously described neutral aminopeptidases with specificity for aromatic residues. The plasma membrane fraction hydrolyzed Tyr-, Trp-, Ala-Pro- and Pro-AP substrates, and also exhibited activity against Phe- and Leu-substrates. Many of the properties of the aminopeptidases, such as pH optima, metal requirements and responses to inhibitors, overlap with those of the previously characterized NPA amidase, suggesting that the latter may represent the combined activities of multiple aminopeptidases.