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PubMed2002

Leucine aminopeptidase during meiotic development.

We found a leucine aminopeptidase (LAP; EC 3.4.11.1) to be abundant in meiotic prophase tissue of a basidiomycete, Coprinus cinereus. After direct purification of the aminopeptidase component from meiocytes, we cloned the gene by degenerate PCR using partial amino-acid sequences of the purified enzyme and 5' and 3' RACE. It was homologous to the eukaryotic leucine aminopeptidase gene. The recombinant protein possesses the characteristic activities of a Coprinus leucine aminopeptidase (CoLAP) with a molecular mass of 52.4 kDa, and forms a homohexamer. Northern blot and spatial distribution analysis by immunohistochemical staining indicated CoLAP to be abundant in meiotic prophase cells and the supporting cells around meiocytes, but scarce in mycelium cells. Interestingly, from zygotene to pachytene, CoLAP was mostly present in supporting cells around meiocytes, but from diplotene onwards, it was plentiful in meiocytes themselves, suggesting that its expression is required to control some of the biochemical events at meiotic prophase. Moreover, the strong expression of CoLAP mRNA immediately after treatment with methyl methanesulfonate in mycelium implies that CoLAP has a role in somatic DNA repair.

Amino Acid Sequence↗

PubMed2002

Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature.

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

Amino Acid Sequence↗

PubMed1991

Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis.

The three-dimensional structures of native bovine lens leucine aminopeptidase (EC 3.4.11.1) and its complex with bestatin, a slow-binding inhibitor, have been solved and exhaustively refined. The mode of binding of bestatin to leucine aminopeptidase may be similar to that of a tetrahedral intermediate that is thought to form during peptide bond hydrolysis. Bestatin binds in the active site with its alpha-amino group and hydroxyl group coordinated to the zinc ion located in the readily exchangeable divalent cation binding site. Its phenylalanyl side chain is stabilized by van der Waals interactions with Met-270, Thr-359, Gly-362, Ala-451, and Met-454, which appear to form a terminal hydrophobic pocket. The leucyl side chain binds in another hydrophobic cleft lined by Asn-330, Ala-333, and Ile-421. Hydrogen bonds involving active site residues Lys-262, Asp-273, Gly-360, and Leu-362 are responsible for stabilizing the backbone nitrogen and oxygen atoms of bestatin. The mode of bestatin inhibition of leucine aminopeptidase is discussed and correlated with biochemical studies of bestatin analogues. In addition, features of a mechanism of catalysis of peptide hydrolysis by leucine aminopeptidase are discussed.

Amino Acid Sequence↗

PubMed2001

A new class of mutants deficient in dodecamerization of aminopeptidase 1 and vacuolar transport.

Vacuolar aminopeptidase 1 is transported to the vacuole by cytoplasmic double-membrane vesicles, the nonclassic Cvt pathway. The cytosolic protein dodecamerizes and is enclosed in a double-membrane vesicle, which is transported to and fuses with the vacuole releasing a single-membrane autophagic body into the vacuolar lumen. This is degraded and the precursor sequence of aminopeptidase 1 is removed. This pathway resembles autophagy, and most proteins identified to function in the Cvt pathway are also required for autophagy and vice versa. The cytosolic precursor protein and the matured vacuolar protein form a homododecameric complex, and only this complex has enzymatic activity. We developed a new genetic screen to isolate mutants in the biogenesis of vacuolar aminopeptidase 1 based on its enzymatic activity. The sensitivity of this assay made it possible for us to search for mutants under conditions where autophagy is down-regulated, and we describe two new mutants defective in the biogenesis pathway of vacuolar aminopeptidase 1. Mutants are defective in dodecamerization of pApe1p and in Cvt vesicle formation. Complex assembly and transport vesicle formation appear to be linked processes. This mechanism can control the potentially harmful cytoplasmic proteolytic activity and could be the driving force for this nonclassic mechanism of vacuolar enzyme transport.

Aminopeptidases↗

PubMed2005

Staphylococcus aureus aminopeptidase S is a founding member of a new peptidase clan.

Staphylococcus aureus aminopeptidase S (AmpS) has been named for its predicted, but experimentally untested, aminopeptidase activity. The enzyme is homologous to biochemically characterized aminopeptidases that contain two cobalt or zinc ions in their active centers, but it is unrelated to all structurally characterized metallopeptidases. Here, we demonstrate AmpS aminopeptidase activity experimentally, and we present the 1.8-A crystal structure of the enzyme. Two metal ions with full occupancy and a third metal ion with low occupancy are present in the active site. A water molecule and Glu-319 serve as bridging ligands to the two metals with full occupancy. One of these metal ions is additionally coordinated by Glu-253 and His-348 and the other by His-381 and Asp-383. In addition, the metals are involved in weak metal-donor interactions to a water molecule and to Tyr-355. In the crystal, AmpS forms a dimer with a large internal cavity. The active sites are located at opposite ends of this internal cavity and are essentially inaccessible from the outside, suggesting that an inactive conformation was crystallized. Because gel filtration and analytical ultracentrifugation data also suggest dimer formation, the problem of substrate access to the active site cavity remains unresolved.

Aminopeptidases↗

PubMed2002

Aminopeptidases in Caenorhabditis elegans and Panagrellus redivivus: detection using peptide and non-peptide substrates.

Aminopeptidase activities were detected in extracts of the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus using the aminoacyl substrate L-alanine-4-nitroanilide. The activities exhibited similarities in Km (C. elegans = 2.22 mM; P. redivivus = 2.09 mM) and specific activity (C. elegans = 1.38 +/- 0.43 mAU min(-1) x g(-1); P. redivivus, 1.23 +/- 0.18m AU min(-1) microg(-1). Each is inhibited competitively by amastatin (C. elegans IC50 = 0.46 microM; P. redivivus IC50 = 15.90 microM) and non-competitively by leuhistin (C. elegans IC50 = 3.00 microM; P. redivivus IC50 = 37.35 microM). The bioactive peptides adipokinetic hormone and substance P decrease the apparent aminopeptidase activities of each extract suggesting that the peptides compete with the Ala-pNA as substrates. With each extract, adipokinetic hormone appeared to be the more effective substrate. Digestion of adipokinetic hormone by C. elegans and P. redivivus extracts in the presence and absence of 1 mM amastatin produced distinct chromatographic profiles that suggest different digestion patterns for the two species. However, amastatin had clear effects on chromatographic profiles from each species indicating that an aminopeptidase is involved in the digestion of the peptide substrates. The data presented indicate that extracts of free-living nematodes are capable of metabolizing peptide hormones, and that this metabolism involves substrate-selective aminopeptidases.

Amino Acids↗

PubMed1996

Prolyl aminopeptidase is also present in Enterobacteriaceae: cloning and sequencing of the Hafnia alvei enzyme-gene and characterization of the expressed enzyme.

The Hafnia alvei prolyl aminopeptidase gene (hpap) was cloned and sequenced, the expressed enzyme (HPAP) was purified to homogeneity and thoroughly characterized. An open reading frame of 1,281 bp was found to code for the enzyme, resulting in a protein of 427 amino acids with a molecular weight of 48,577. HPAP resembles the Aeromonas sobria enzyme, having 45% identity and the same distinctive properties with respect to size and substrate specificities. Both enzyme show similar chromatographic behavior, and HPAP could be purified following the procedure previously described for the Aeromonas enzyme. HPAP was found to be resistant to diisopropylphosphofluoridate as are most of the prolyl aminopeptidases hitherto described. In spite of this similarity, no inhibition by 1 mM p-chloromercuribenzoate solution could be detected. Significant inhibition was, however, observed when the enzyme was incubated with 3,4-dichloroisocoumarin. This study confirms the presence of two types of prolyl aminopeptidases, of which the Hafnia and Aeromonas enzymes constitute one group and the Bacillus, Neisseria, and Lactobacillus enzymes the other, and describes the cloning of the first prolyl aminopeptidase gene from an Enterobacteriaceae.

Aeromonas↗

PubMed1989

Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme.

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.

Amino Acids↗

PubMed1980

Purification and properties of aminopeptidase and arylamidase from human liver.

Aminopeptidase and arylamidase from human liver were purified 1,580- and 1,130-fold, respectively, by ammonium sulfate fractionation, TEAE-cellulose and hydroxylapatite column chromatographies, and Sephadex G-200 gel filtration. The purified preparations appeared to be homogeneous on polyacrylamide gel electrophoresis. The molecular weights of aminopeptidase and arylamidase were determined to be 280,000 and 170,000, respectively, by gel filtration on Sephadex G-200. Aminopeptidase showed marked specificities for amino acid amides, such as L-leucineamide, L-phenylalanineamide, and L-methionineamide. On the other hand, arylamidase showed specificities for amino acid beta-naphylamides, such as L-alanyl-beta-naphthylamide and L-leucyl-beta-naphthylamide, but it also hydrolyzed amino acid amides to a considerable extent. Immunological studies using antibodies to purified aminopeptidase and arylamidase showed that the antigenicities of these two enzymes were different. Neuraminidase treatment of the purified arylamidase changed its isoelectric point from 3.25 to 4.95. Various properties (Km values, optimum pH, substrate specificities, and antigenicity) of the arylamidase were not changed by neuraminidase treatment, but the enzyme became thermo-labile.

Aminopeptidases↗

PubMed2004

Collagen production in cardiac fibroblasts during inhibition of angiotensin-converting enzyme and aminopeptidases.

OBJECTIVE: To determine whether lisinopril, an angiotensin-converting enzyme (ACE) inhibitor, and bestatin, an aminopeptidase inhibitor with broad specificity, could affect collagen production in control and transforming growth factor (TGF)-beta1-treated cardiac fibroblasts. DESIGN AND METHODS: Cardiac fibroblasts from passage 2 from normal male adult rats were cultured to confluency, incubated with or without 600 pmol/l TGF-beta1 for 2 days in serum-free Dulbecco's modified Eagle's medium and then incubated with the test products (lisinopril or bestatin) for 1 day in this medium with added ascorbic acid, beta-aminoproprionitrile and tritiated proline. Soluble collagen was measured in the conditioned medium and non-soluble collagen in the cell layer. ACE activity was measured fluorimetrically with hippuryl-histidyl-leucine as substrate, and DNA with the bisbenzimide dye, Hoechst 33,258. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine-p-nitroanilide. RESULTS: Lisinopril dose-dependently reduced ACE activity in control and TGF-beta1-treated cardiac fibroblasts. Bestatin inhibited the basal and TGF-beta1-stimulated aminopeptidase activity in a concentration-dependent manner. Lisinopril (10 micromol/l) decreased (P < 0.05) the production of soluble and non-soluble collagen in control cardiac fibroblasts. TGF-beta1 (600 pmol/l) increased (P < 0.05) the production of soluble and non-soluble collagen, and this effect was decreased (P < 0.05) by lisinopril. Bestatin (100 micromol/l) reduced (P < 0.01) the production of soluble collagen in control and TGF-beta1-treated cardiac fibroblasts, but did not affect the production of non-soluble collagen in these cells. CONCLUSIONS: Our data suggest that ACE and aminopeptidases are involved in the basal and TGF-beta1-stimulated production of collagen in adult rat cardiac fibroblasts in culture.

Aminopeptidases↗

PubMed1996

A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171.

An aminopeptidase with a very broad substrate specificity was purified to homogeneity from Lactobacillus helveticus SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide- and nitroanilide-substituted amino acids, as well as several di-, tri- and oligopeptides. It also exhibited significant proline-iminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl-p-nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a Vmax of 2.5 mumol min-1 (mg protein)-1 while lysyl-p-nitroanilide was hydrolysed with a high affinity [Km 0.003 mM; Vmax 37.5 mumol min-1 (mg protein)-1]. The aminopeptidase activity, which was optimal between pH 6.0 and 8.0 and at 50 degrees C, was very stable at 30 degrees C for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with beta-mercaptoethanol, while Co2+ and Mn2+ restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from Lactococcus lactis and Lb. helveticus clearly showed that both aminopeptidases are at least immunologically different from each other.

Amino Acid Sequence↗

PubMed1995

Capnocytophaga gingivalis aminopeptidase: a potential virulence factor.

The production and properties of an aminopeptidase from Capnocytophaga gingivalis were studied. C. gingivalis was grown in continuous culture over a range of dilution rates and the cell-bound and extracellular levels of aminopeptidase and trypsin-like protease (TLPase) measured. At high growth rates (0.6 mu rel) TLPase specific activity was low and found exclusively as cell-bound activity; at low growth rates (0.0375 mu rel), specific activity was high and 26% was found as extracellular activity. In contrast, aminopeptidase specific activity was highest at 0.3 mu rel and the ratio of cell-bound to extracellular activity was relatively constant at all growth rates. Only about 5% of the total activity was extracellular. The aminopeptidase, which has a wide specificity towards artificial substrates, was purified to homogeneity, as judged by SDS-PAGE, from the supernatant fluid of cells grown in continuous culture in a tryptone/glucose/thiamine medium. The enzyme has a molecular mass of 61 kDa, a pl of 6.3, a pH optimum close to 7.5 and showed a requirement for magnesium or calcium ions. The N-terminal sequence of the first 10 amino acids (Asp-Val-Asn-Met-Leu-Trp-Tyr-Val-x-Arg...) showed no similarity to any published sequence. This enzyme in its cell-bound or extracellular form may be important in the nutrition and pathogenesis of C. gingivalis in the human oral cavity.

Amino Acid Sequence↗

PubMed2005

Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum.

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58-65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

Amino Acid Sequence↗

PubMed2000

Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 A resolution.

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.

Aeromonas↗

PubMed2005

Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis.

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.

Aminopeptidases↗

PubMed1979

Soluble di- and aminopeptidases in Escherichia K-12. Dispensible enzymes.

As part of a study of the peptidase content of Escherichia coli K-12, two peptidase-deficient amino acid auxotrophs isolated and characterized by Miller as pepD- (strain CM17) and pepD- pepN- pepA- pepB- pepQ- (strain CM89) were examined for the presence of several peptidases previously obtained from strain K-12 in this laboratory. The soluble fraction of each mutant was found to lack the broad-specificity strain K-12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K-12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide-specific aminopeptidase TP. Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome-bound aminopeptidase I of strain K-12. Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD-, pepB-, pepN-, and pepA- mutations, respectively, the reason for the loss of L activity remains uncertain. Grown responses of strain CM89 in liquid media containing di- or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides. In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K-12 and produced smaller cell-yields than those produced by strain K-12.

Amino Acid Sequence↗

PubMed1985

Distinct behavior of beta-endorphin and corticotropin toward leucine aminopeptidase action.

Reactions of human beta-endorphin, corticotropin and their synthetic analogs with leucine aminopeptidase have been investigated. The results confirmed previous findings that beta-endorphin is resistant to the aminopeptidase action whereas corticotropin is not. Beta-endorphin-(1-5) is completely digested by the enzyme while beta-endorphin-(1-17) is resistant. In contrast, the NH2-terminal 7 residues in corticotropin are removed readily by leucine aminopeptidase. This is confirmed by the observation that human corticotropin-(7-38) is not hydrolyzed by the enzyme. This contrasting behavior of the two hormones toward leucine aminopeptidase may be related to differences in their conformational structures.

Adrenocorticotropic Hormone↗

PubMed1976

An extracellular aminopeptidase from Clostridium histolyticum.

An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.

Amino Acids↗