Leucine aminopeptidase (bovine lens). Effect of pH on the relative binding of Zn2+ and Mg2+ to and on activation of the enzyme.

Incubation of leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with various concentrations of Mg2+ at various pH values in 1 M KCl and 0.155 M trimethylamine-HCl at 37 degrees confirms that Mg2+ competes with Zn2+ for binding only 1 site per 54,000-dalton subunit. The ratio of the apparent association constants (1KZn:1KMg = 1KZn/Mg) at this site (site 1) was estimated to be 20,720 at pH 8.16, 10,570 at pH 8.44, 3,590 at pH 8.78, and 660 AT PH 9.14. The decrease in values of 1KZn/Mg with increasing pH in the activation of leucine aminopeptidase by Mg2+ is attributed to the lowering of the free Zn2+ concentration relative to that of free Mg2+ caused by the formation of ZnOH+ and Zn(OH)2 complexes with increasing OH- concentration. When corrections are made for the binding of Zn2+ by OH- ions, the pH-independent ratio of association constants (1KZn:1KMg = 1KZn/Mg) for the relative binding of Zn2+ and Mg2+ at site 1 of leucine aminopeptidase in 29,800. From the effect of pH on the relative binding constant, a value (beta2) for the product of the two stepwise association constants for the formation of Zn(OH)2 from Zn2+ and OH- (Zn2+ + OH- in equilibrium ZnOH+; ZnOH+ + OH- in equilibrium Zn(OH)2) was estimated to be 4.42 X 10(10) M-2 at 37 degrees. Values of Km at pH 7.5 AND 30 degrees with L-leucine p-nitroanilide as substrate in the presence of 0.01 M NaHCO3 are 4.13 and 2.01 mM for the zinc-zinc and magnesium-zinc enzymes, respectively. Values for Vmax are 0.2 and 2.49 mumol/min/mg, respectively.     

The yeast aminopeptidase Y

A metal-dependent aminopeptidase (EC 3.4.11.-), designated APase Y, has been purified to homogeneity by conventional methods. The enzyme is composed of a single polypeptide chain with molecular mass of 102 kilodaltons, estimated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis, with a blocked N-terminal amino acid. It possesses neither endopeptidase nor carboxypeptidase activity and is strongly inhibited by metal-chelating agents, Zn2+, and the protein inhibitor from Neurospora crassa. APase Y is insensitive to Cl anions, S--S reducing reagents, serine protease inhibitors, and the peptidase inhibitor benzamidine. Co2+, Hg2+, and p-chloromercuribenzoate can activate the enzyme up to 22, 20, and 55%, respectively. The holoenzyme is resistant to yeast endopeptidases A, B, and Y, whereas the apoenzyme (obtained after treatment with chelators) is susceptible to the serine endopeptidases B and Y. The enzyme catalyzes hydrolysis of most L peptides possessing free alpha-amino (or imino) group by stepwise removal of N-terminal residue. Peptides with L-leucine at the N terminus are cleaved preferentially. The enzyme is unable to catalyze hydrolysis of X--Pro type peptide bonds, and inefficiently hydrolyzes bonds between Asp--X and Glu--X. L-leucine p-nitroanilide hydrolyzes optimally at pH 8.2 with a Km value of 1 mM. The purified enzyme is stable during storage in 0.05 M phosphate buffer, pH 6.7, containing 40-50% glycerol, at -20 degrees C.     

Purification and characterization of aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinylglycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidsase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. Alpha-glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26

An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. Methylene-blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino-acid analysis indicated that this photooxidative loss of activity corresponds to the modification of one histidine residue per monomer of protein.     

Interaction of chloride with yeast aminopeptidase I. Equilibrium binding studies

The effect of chloride on metal binding by yeast aminopeptidase I, as well as the binding of chloride to various enzyme forms were studied by means of a micro-centrifugation technique using radioactive 36Cl- as a ligand. Chloride did not significantly alter the binding of activating Zn2+, or binding of Co2+ to the essential metal sites. Both the native Zn2+ enzyme and Co2+-substituted aminopeptidase I bind stoichiometric amounts of C1- (1 Cl-/subunit) with apparent dissociation constants of 0.1-0.2 mM. Additional Cl- was bound at higher concentrations. In contrast to the metal-containing enzyme forms the apoenzyme did not express the high-affinity chloride binding site.     

Characterisation of aminopeptidase activity in scab mites (Psoroptes spp.)

Soluble and membrane-bound aminopeptidase activities were demonstrated in extracts of P. cuniculi (Delafond). Leucine aminopeptidase (LAP) activity in the soluble fraction of P. cuniculi extracts displayed substrate preference for amino acid derivatives with terminal leucine and methionine over those with acidic, basic or heterocyclic groups. P. cuniculi LAP was inhibited by leucinethiol (IC(50) = 1.4 +/- 0.4 nM), bestatin (IC(50) = 3.9 +/- 1.7 microM), Arphamenine A (IC(50) = 0.37 +/- 0.03 mM) the chelating agent 1,10-phenanthroline (IC(50) = 2.3 +/- 0.5 mM), Zn(2+), Cu(2+) Ni(2+), and Co(2+), and activated by Mn(2+) and Mg(2+). The LAP activity was visualised as a single major band after electrophoresis on native gels and eluted from a size exclusion column as a single major peak representing a molecular mass range of 85-116 kDa. Degenerate oligonucleotide primers were used to amplify short fragments of genomic DNA containing nucleotide sequence coding for the cation-binding motifs of the co-catalytic Zn(2+) binding domains of dizinc leucine aminopeptidases in both P. cuniculi and P.ovis (Hering). The major soluble aminopeptidase from these mites therefore displays most of the characteristics associated with typical cytosolic leucine aminopeptidases belonging to the M17 family of metalloproteinases.     

Inhibition of aminopeptidase M by alkyl D-cysteinates

Ethyl D-cysteinate is a potent competitive inhibitor (Ki = 3.5 x 10(-7) M) of aminopeptidase M. D-cysteine and ethyl L-cysteinate inhibit more than two orders of magnitude less effectively. Inhibition studies on several n-alkyl esters of D-cysteine reveal an optimum at the n-butyl ester (Ki = 1.8 x 10(-7) M). The results are consistent with the hypothesis that the thiol group coordinates to Zn+2 at the active site and the alkyl group occupies the hydrophobic binding site for the side chain of the amino-terminal residue of substrates. Cytosolic leucine aminopeptidase is not significantly inhibited by ethyl D-cysteinate.     

Characteristics of aminopeptidase activity of female housefly reproductive accessory glands

Proteolytic activity was detected in crude extracts of female reproductive accessory glands and the following characteristics of the principal aminopeptidase activities were determined: substrate specificity, pH optima, molecular weights, and effects of inorganic salts. The greatest aminopeptidase activities were found with the β-naphthylamides of: alanine at pH 7.5 and 9.5, leucine at pH 8.0, and methionine at pH 6.5. The methionine-specific activity in the crude extract was stimulated 3 times by 100 mM MgCl₂, CaCl₂, NaCl, or KCl. Inhibition was noted, and ID₅₀ was determined for each of the other principal substrates with the following salts: CdCl₂, CaCl₂, ZnCl₂, HgCl₂, MgCl₂, and MnCl₂. Molecular weights, estimated on Sephadex G-200 and on Sepharose-6B, were found to be around 210 000 for each of these principal aminopeptidase activities in the crude extract.     

Mammalian lens dipeptidyl aminopeptidase III

An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K_m for this substrate was estimated to be 2.83 × 10^?5M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.     

Zinc potentiation of androgen receptor binding to nuclei in vitro

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 microM ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl, and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 microM total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added at a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; however, in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 X 10(-13) mol of receptor sites/mg of DNA, corresponding to approximately 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10(13) M-1 was approximated. Pyridoxal 5'-phosphate (less than or equal to 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 10 mM pyridoxal 5'-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).     

An activated by cobalt alkaline aminopeptidase from Bacillus mycoides

An intracellular arginine--specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of -50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-beta-naphthylamide as a substrate was at 37 degrees C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co2+ ions activated the enzyme, while Zn2+, Cu2+, Hg2+ and Mn2+ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to AmpS of Bacillus cereus and AP II of B. thuringensis.     

Spectral and kinetic studies of metal-substituted Aeromonas aminopeptidase: nonidentical, interacting metal-binding sites

Apoenzyme prepared by removal of the 2 mol of Zn2+/mol from Aeromonas aminopeptidase is inactive. Addition of Zn2+ reactivates it completely, and reconstitution with Co2+, Ni2+, or Cu2+ results in a 5.0-, 9.8-, and 10-fold more active enzyme than native aminopeptidase, respectively. Equilibrium dialysis and spectral titration experiments with Co2+ confirm the stoichiometry of 2 mol of metal/mol. The addition of only 1 mol of metal/mol completely restores activity characteristic of the particular metal. Interaction between the two sites, however, causes hyperactivation; thus, addition of 1 mol of Zn2+/mol subsequent to 1 mol of Co2+, Ni2+, or Cu2+ per mole increases activity 3.2-, 42-, or 59-fold, respectively. The cobalt absorption spectrum has a peak of 527 nm with a molar absorptivity of 53 M-1 cm-1 for 1 mol of cobalt/mol, which increases to 82 M-1 cm-1 for a second cobalt atom and is unchanged by further addition of Co2+. Circular dichroic (CD) and magnetic CD spectra indicate that the first Co2+ binding site is tetrahedral-like and that the second is octahedral-like. Stoichiometric quantities of 1-butylboronic acid, a transition-state analogue inhibitor of the enzyme [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322], profoundly affects absorption, CD, and MCD spectra, but n-valeramide, a substrate analogue inhibitor, has no effect. These findings suggest that the tetrahedral-like site is catalytic and the other octahedral-like site is regulatory or structural.     

Proteases of swine small intestine enterocytes. Kinetics of substrate hydrolysis and inhibition of aminopeptidase N from brush border vesicles

The kinetics of hydrolysis of L-leucine p-nitroanilide and some p-nitrophenylalanine dipeptides by vesicular aminopeptidase N from the porcine small intestine brush border membrane was studied. It was shown that the catalytic properties of the vesicular enzyme are very similar to those known for its solubilized counterpart. Both enzymes are inhibited by o-phenanthroline, ZnCl2 and puromycin with Ki = 10(-5)-10(-6) M. The data obtained offer new possibilities for investigating the role of aminopeptidase N in the amino acid and peptide transport across the enterocyte membrane.     

Albumins activate peptide hydrolysis by the bifunctional enzyme LTA4 hydrolase/aminopeptidase.

Albumins from several species activated the bifunctional, Zn2+ metalloenzyme amino-peptidase/leukotriene A4 hydrolase (EC 3.3.2.6). Bovine serum albumin, 1 mg/mL, increased hydrolysis of L-proline-p-nitroanilide and leucine-enkephalin by 12-fold and 7-fold, respectively. The apparent Km for L-proline-p-nitroanilide was inversely proportional to the albumin concentration from 0 to 1 mg/mL, declining from 9.4 to 0.7 mM without an appreciable change in apparent Vmax. These data imply a random activation process in which the enzyme-activator complex is catalytically dominant. Hill plots indicated a 1:1 stoichiometric relationship between albumin and enzyme. Secondary plots of slope versus the reciprocal of albumin concentration indicated that it binds to the enzyme with an affinity constant of 0.9 microM. The pH optimum of the nonactivated enzyme occurred at pH 8; the albumin-activated enzyme had an optimum near pH 7. Neither ultrafiltration nor dialysis of albumin altered its activating effect, but boiling abolished it. Albumin did not affect other cytosolic or microsomal leucine aminopeptidases, or gamma-glutamyltransferase. Albumin functions as a nonessential activator, since enzymatic activity was always detectable in its absence. Chloride ions, which activate other Zn2+ metalloenzymes, also activated leukotriene A4 hydrolase/aminopeptidase with an EC50 = 50 mM, increasing its initial velocity 2.2-fold in the absence of albumin. Zn2+ activated the enzyme, increasing its apparent Vmax but not its apparent Km, suggesting it replaced Zn2+ lost from the active site, especially at acidic pH. At concentrations greater than 30-50 microM, Zn2+ was inhibitory. Albumin mitigated the effect of chloride, but not the effect of Zn2+ or that of the competitive inhibitor, captopril.(ABSTRACT TRUNCATED AT 250 WORDS)     

An inexpensive device for sample stirring in VSU Zeiss spectrophotometers. Its use for kinetic measurements and active site titrations with immobilized enzymes.

The construction of a simple and effective sample stirring device for commercial spectrophotometers and its use for continuous kinetic measurements and active site titrations with immobilized enzymes is described. Sepharose-bound leucine aminopeptidase and trypsin were selected as model enzymes to test the performance of the magnetic stirring equipment. Kinetic parameters of insolubilized leucine aminopeptidase using L-leucine p-nitroanilide as substrate and the catalytic site concentration of matris-bound trypsin using p-nitrophenyl p'-guanidinobenzoate as active site titrant could be evaluated without significant interference from the turbidity of the stirred Sepharose suspension. The problem of grinding of the support material could be overcome. Both unbound native and carrier-fixed enzyme may be reacted under identical conditions with similar convenience and sensitivity.     

Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase).

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.     

Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli

The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed.     

Purification and characterization of an aminopeptidase from sperm of the sea urchin, Strongylocentrotus intermedius. Ca2(+)-dependent substrate specificity as a novel feature of the enzyme

An aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (Mr = 110,000) with an isoelectric point of 5.2 and shows the highest activity in a pH range between 7.0 and 7.5. Ni2+, Cu2+, Zn2+, and Hg2+, as well as 1,10-phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Ca2+, Mn2+, Mg2+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as arginine-MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolysis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single active site of the enzyme. Furthermore, the enzyme showed an identical Ki value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.     

Aminopeptidase from the skeletal muscle of fresh water fish Tilapia mossambica

An aminopeptidase from the skeletal muscle of fish, Tilapia mossambica, was partially purified to 96-fold using salt precipitation, ion-exchange chromatography and molecular sieve chromatography. The enzyme showed optimum activity between pH 6.5-7.5 at 43 degrees C and Vmax and Km of 14.36 units/mg and 0.059 mM respectively with alanine beta-naphthylamide as the substrate. The aminopeptidase having a molecular weight of 305 kDa was activated by sulphydryl compounds and Co2+ and inhibited by bestatin, puromycin and metal chelators. Inhibition caused by metal chelators could be reversed by the addition of Co2+. Inclusion of L-amino acids, particularly isoleucine and leucine, in the assay medium caused inhibition of the enzyme activity. Substrate specificity together with inhibition and activation pattern indicated that the enzyme is alanine aminopeptidase.