L-leucine P-nitroanilide Research Articles

Kinetics and conformational stability studies of recombinant leucine aminopeptidase

Leucine aminopeptidase from Vibrio proteolyticus is a broad specificity N-terminal aminopeptidase that is widely used in pharmaceutical processes where the removal of N-terminal residues in recombinant proteins is required. We previously reported the expression of a heterologous construction of the mature protein fused to a 6-histidine tag that presents a reasonable refolding rate for its use at industrial level. Here, we investigate this recombinant leucine aminopeptidase (rLAP) to explain the gain of activity observed when incubated at 37°C after its production. Unfolding transitions of rLAP as a function of urea concentration were monitored by circular dichroism (CD) and fluorescence (FL) spectroscopy exhibiting single transitions by both techniques. Free energy change for unfolding measured by CD and FL spectroscopy are 2.8±0.4 and 3.7±0.4kcalmol−1, respectively. Thermal stability conformation of rLAP is 2.6±0.1 and 6.1kcalmol−1 for CD and Nano-Differential Scanning Calorimetry (Nano-DSC), respectively. Enzyme activity was assessed with l-leucine-p-nitroanilide (L-pNA) as substrate. The catalytic efficiency was 3.87±0.10min−1μM−1 at 37°C and pH 8.0. Kinetic and conformation studies show differences between the enzyme native and rLAP; however rLAP is selective and specific to remove N-terminal groups from amino acids.

Endoprotease and Exopeptidase Activities in the Hepatopancreas of the Cuttlefish Sepia officinalis, the Squid Todarodes pacificus, and the Octopus Octopus vulgaris Cuvier

This study examined and compared the exopeptidase and endoprotease activities of the hepatopancreas (HP) of cuttlefish, squid, and octopus species. The protein concentration in crude extract (CE) from octopus HP was 3,940 mg/100 g, lower than those in CEs from squid HP (4,157 mg/100 g) and cuttlefish HP (5,940 mg/100 g). With azocasein of pH 6 as a substrate, the total activity in HP CE of octopus was 31,000 U, lower than the values for cuttlefish (57,000 U) and squid (69,000 U). Regardless of sample type, the total activities of the CEs with azocasein as the substrate were higher at pH 6 (31,000-69,000 U) than at pH 9 (19,000-34,000 U). With L-leucine-p-nitroanilide (LeuPNA) of pH 6 as the substrate, the total activity of the HP CE from octopus was 138,000 U, higher than values from both cuttlefish HP (72,000 U) and squid HP (63,000 U). Regardless of sample type, the total activities of the CEs with LeuPNA as the substrate were higher at pH 6 (63,000-138,000 U) than at pH 9 (41,000-122,000 U). With LeuPNA as the substrate, the total activities of the CEs from octopus HP and cuttlefish HP were higher at pH 6 than at pH 9. However, there was no difference in total activity between pH 6 and 9 for squid HP CE with LeuPNA as the substrate. These results suggest that the octopus HP is superior to the cuttlefish HP and squid HP as a potential resource for extracting exopeptidases. Exopeptidases from octopus HP have potential industrial applications and their use might aid in reducing pollution related to the octopus industry.

The leucine aminopeptidase of Staphylococcus aureus is secreted and contributes to biofilm formation

Staphylococcus aureus has emerged as a major drug-resistant pathogen in hospital- and community-acquired infections. Leucine aminopeptidase (LAP) is known to be essential for survival of the bacteria; however the LAP of S. aureus has not been extensively characterized. In this study, we report a detailed characterization of the S. aureus LAP. LAP from S. aureus was cloned, purified, and further biochemically characterized. The expression of LAP was analyzed by Western blotting. Growth and biofilm formation were analyzed spectrophotometrically. LAP was cloned from S. aureus and expressed as a 55 kDa protein, whereas the molecular weight of the native protein is approximately 600 kDa. LAP showed amidolytic activity against l-leucine p-nitroanilide. Optimal activity was observed at pH 8.5 and 37°C with a V(max) of 2500μmol/min/mg protein. LAP enzymatic activity was inhibited by ion chelators and enhanced by divalent metal ions, specifically Ni. LAP is secreted by laboratory as well as clinical strains. Bestatin, an inhibitor of LAP, inhibits S. aureus growth and biofilm formation. To our knowledge, this is the first detailed characterization of LAP from S. aureus and suggests its importance in survival and pathogenesis.

Preparation and catalytic properties of trypsin immobilized on cryogels of polyvinyl alcohol

Commercial preparations of trypsin, varying in activity, were immobilized on a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In organic media, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-arginine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.

Kinetic, Spectroscopic, and X-ray Crystallographic Characterization of the Functional E151H Aminopeptidase from Aeromonas proteolytica,

Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the D-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a kcat value of 2.0 min(-1), which is over 2000 times slower than r AAP (4380 min(-1)). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV-vis and EPR spectra of CoII-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters kcat and K(m) for the hydrolysis of L-leucine p-nitroanilide (L-pNA) revealed a change in an ionization constant in the enzyme-substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 A resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear ZnII active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely shuttles a proton to the leaving group of the substrate.

ZnII Complex with a Phenanthroline‐Containing Macrocycle as Receptor for Amino Acids and Dipeptides − Hydrolysis of an Activated Peptide Bond

AbstractThe interaction between several L‐amino acids and dipeptides and the ZnII complex with ligand L, a macrocycle containing a triamine chain linking the 2,9 positions of a phenanthroline moiety, has been studied by means of potentiometric and 1H NMR spectroscopic measurements in aqueous solutions. In the [ZnL]2+ complex, the metal ion displays an unsaturated coordination environment and the metal can act as a binding site for these substrates. Amino acids and dipeptides in their neutral (zwitterionic) form bind to ZnII through the carboxylate function and, when in their anionic form, through the amine group. In this case the carbonyl of the amide function is probably also involved in metal coordination. Amino acids containing aromatic pendants form the most stable complexes, due to hydrophobic and/or π‐stacking interactions between the aromatic subunits of the substrates and the phenanthroline moiety of the metal receptor. The hydrolytic properties of the ZnII complex with L, involving the cleavage of the peptide bond, have been tested by using L‐leucine‐p‐nitroanilide (LNA), which contains an activated peptide bond. This substrate forms stable [ZnL(LNA)]2+ and [ZnL(OH)(LNA)]+ complexes. The formation of the [ZnL(OH)(LNA)]+ complex is followed by LNA hydrolysis, through a nucleophilic attack of the Zn−OH function on the peptide bond. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Some Properties of Proteolytic Enzymes and Storage Proteins in Recalcitrant and Orthodox Seeds of Araucaria

Araucaria bidwillii Hook. and Araucaria cunninghamii Don D. are two species of conifers whose seeds belong to different physiological categories: A. bidwillii seeds are recalcitrant, while A. cunninghamii seeds are orthodox. The extraction of enzymes and storage proteins was carried out from A. bidwillii and A. cunninghamii megagametophytes. The endopeptidase activities of both species were assayed with azocasein and with haemoglobin; the exopeptidase activities were detected by various N-carbobenzyloxy-dipeptides and L-leucine p-nitroanilide. The use of appropriate proteinase inhibitors, i.e. pepstatin A, ethylenediaminetetraacetic acid and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, showed the presence of aspartic and metallo proteinases and the absence of the cysteine ones both in A. bidwillii and in A. cunninghamii ungerminated seeds. Since the results do not show differences between the types of enzymes in the ungerminated araucarian seeds and those present in some ungerminated angiosperm seeds (barley, wheat, maize, rice, buckwheat), we conclude that their physiological role is similar. The electrophoretical analyses of soluble and insoluble storage proteins of A. cunninghamii showed patterns similar to those found in other gymnosperms, while the storage protein patterns of A. bidwillii seeds were rather atypical.

Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N

The purpose of this study was to evaluate the potential of polycarbophil–cysteine conjugates (PCP–Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 μM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP–Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP–Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 ± 4 and 60 ± 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP–Cys (0.25% w/v, pH 6) 11 ± 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1907–1914, 2001

Aminopeptidase-N from the Helicoverpa armigera (Hubner) brush border membrane vesicles as a receptor of Bacillus thuringiensis crylac delta-endotoxin.

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type δ-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2′-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of δ-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60–77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

A new technique for detecting oxytocinase activity in electrophoresis gels.

A sensitive staining method for the detection of oxytocinase (EC 3.4.11.3) activity in electrophoresis gels has been described. The method is based on the enzymatic release of p-nitroaniline (PNA) from two specific synthetic oxytocinase substrates, S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN), respectively. The PNA is then diazotized with sodium nitrite and subsequently coupled to a chromogen, N-(1-naphthyl)-ethylenediamine dihydrochloride (NED) to produce a deep pink/magenta colored azo-dye at the site of oxytocinase activity.

Oxytocinase Activity in Human Spermatozoa and Its Possible Role in Fertilization

Human spermatozoa were extracted with 1% Triton X-100 and analyzed for oxytocinase (E.C. 3.4.11.3) activity by means of two synthetic peptides. S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN), separately as substrates. The specific activity (mean +/- SD) of this proteolytic enzyme at Vmax in eight different extracts was 0.150 +/- 0.072 and 1.392 +/- 0.602 mIU/10(6) cells using BCN and LN, respectively. The enzyme showed optimal activity at pH 7.2 when BCN was the substrate, and at pH 7.4 with LN as the substrate. Spermatozoal oxytocinase activity may be involved directly or indirectly in the reproductive mechanisms leading to sperm acrosome reaction and fertilization.

Fractionation and characterization of oxytocinases in human semen.

Two isoenzymes of oxytocinase activity were fractionated from human seminal plasma by acrylamide-agarose gel chromatography and partly characterized using S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) separately as substrates. These isoenzymes appeared to be metallo-aminopeptidases with different elution volumes (90 ml and 150 ml), apparent molecular weights (unknown value and 300,000) and pH optima (6.8 and 7.0 with BCN and 7.2 and 7.4 with LN), but with similar substrate affinity and thermal sensitivity, and susceptibility to EDTA, divalent metal ions, L-methionine, polypeptide hormones and prostaglandins. A comparison of the enzymic properties with pregnancy-associated oxytocinases suggests that seminal oxytocinases are related more closely to amniotic fluid isoenzymes than to pregnancy serum, placental and uterine isoenzymes.

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.

Proteinases of Legionella: phenylalanineaminopeptidase of L. pneumophila.

Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella pneumophila by affinity chromatography on O-tert-butyl-L-threonyl-L-phenylalanyl-L-prolylglycyl-aminosilo chrom and by gel-filtration; a 401-fold purification with a yield of 18% was achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a pI of 5.8. It was stable at pH 7-9 and had an activity optimum in the range of pH 8-9.5 with L-phenylalanine p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound, substantially lower towards L-leucine p-nitroanilide and only marginal towards other p-nitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine proteinase were also detected in L. pneumophila culture filtrate.

Multimolecular forms of oxytocinase activity in the human uterus and their inhibition by prostaglandins and cyclic GMP

Oxytocinase (EC 3.4.11.3) activity was measured in uterine homogenates from pregnant and non-pregnant women using S-benzyl-L-cysteine-pnitroanilide (BCN) and L-leucine-p-nitroanilide (LN) separately as substrates. There was no significant difference between the specific activity of the enzyme in the pregnant and non-pregnant uterus. The uterine homogenates were also subjected to gel filtration on Ultrogel AcA 22 column and two hydrolyzing activity peaks from the pregnant uterus (PUI, PUII) and three from the non-pregnant uterus (UI, UII, hydrolysis of the two substrates by the enzyme peaks was similar, the hydrolysis of LN was greater than that of BCN. PUII, UII and UIII showed higher affinity for the substrates than PUI and UI, and the affinity of all isoenzymes was significantly greater for LN than BCN. These uterine oxytocinases appeared to be metallo-aminopeptidases and were potently inhibited by Cu , Zn 2+ and prostaglandins (PGs). Cyclic GMP (cGMP) and its 8-bromo derivative, however, inhibited only PUI and UI. The divalent cations and PGs were equipotent against the hydrolysis of both substrates, but cGMPs were more effective against BCN hydrolysis. All of these inhibitions depended on the pH of the incubation medium and types of the inhibition by PGs and cGMPs were mixed and uncompetitive respectively. The implications of these findings in relation to human parturition have been discussed.

Selective inhibition of multimolecular forms of human serum and placental oxytocinase activity by prostaglandins and cyclic GMP

Ultrogel acrylamide-agarose chromatography was employed for fractionation of oxytocinase isoenzymes from serum of pregnant women and from human placenta. Using S-benzyl-L-cysteine-p-nitroanilide (BCN) and L-leucine-p-nitroanilide (LN) as substrates, three activity peaks (PI, PII, PIII) from placenta, and one peak (SI) from serum were identified. SI coincided with PII, and with all isoenzymes the hydrolysis of LN was greater than that of BCN. Prostaglandins E 1, E 2 and F 2α inhibited all oxytocinases, more potently the hydrolysis of LN than BCN and at pH 6.2 than at pH 6.8. Although cyclic GMP and its 8-bromo derivative similarly inhibited these isoenzymes except PIII, they were considerably more effective against the hydrolysis of BCN than LN.

Purification and properties of an aminopeptidase from buckwheat seed.

An aminopeptidase was purified from buckwheat seed by using affinity chromatography, ionexchange chromatography and chromatofocusing. The enzyme had a molecular weight of 37, 000 as determined by gel filtration. The aminopeptidase activity, determined with L-leucine-p-nitroanilide (Leu-PNA) as the substrate, exhibited a pH optimum of 7.2. The Km value for Leu-PNAwas 140 μM. The preferred substrates were L-leucine-β-naphtylamide and Leu-pNA, although there was also high activity against L-leucyl-L-alanine and L-leucinamide. Thiol antagonists were found to be potent inhibitors against the enzyme. The enzymeexhibited less or no sensitivity to the endogeneous proteinase inhibitors, benzamidine and TPCK.

Alpha-aminoaldehydes: transition state analogue inhibitors of leucine aminopeptidase.

L-Leucinal, prepared by enzymatic oxidation of L-leucinol with alcohol dehydrogenase, is found to be a very strong competitive inhibitor of porcine kidney aminopeptidases. For the enzyme from kidney microsomes acting on L-leucine p-nitroanilide (Km = 5.2 x 10(-4) M), for Ki for L-leucinal was 7.6 x 10(-7) M at pH 7.2 and 25 degrees C. For the enzyme from kidney cytosol acting on L-leucine p-nitroanilide (Km = 7.7 x 10(-4) M), Ki for L-leucinal was 6 x 10(-8) M; Ki for glycinal (analogous to glycine derivatives that are poor substrates) was 6.8 x 10(-4) M. In dilute aqueous solution, leucinal exists in unfavorable equilibrium with its covalent hydrate, whose concentration exceeds that of the free aldehyde by a factor of 40. The affinity of the enzyme for the free aldehyde is correspondingly greater than its Ki values would suggest, exceeding the apparent affinity of the substrate by a factor of about 10(6). A comparison of binding affinities suggests that L-leucinal forms an inhibitory complex analogous in structure to unstable intermediates and substrate transformation by leucine aminopeptidase, and strengthens the likelihood that this enzyme may act by a double-displacement mechanism.

PH dependent inhibition of serum oxytocinase activity by prostaglandins and cyclic GMP

The effect of pH in the range 6.2 to 7.7 on the inhibition of serum oxytocinase (EC 3.4.11.3) activity by various compounds was studied using S-benzyl-L-cysteine-p-nitroanilide (BCN) as substrate. Prostaglandins E 1, E 2, F 2α, 8-bromo-cGMP, cGMP, indomethacin and polyphloretin phosphate (PPP) produced a dose related pH dependent inhibition of serum oxytocinase. Their effect was maximum at pH 6.2 and minimum at pH 7.7. Hypertonic urea and saline also caused pH dependent inhibition; saline being most active at pH 6.2, and urea at pH 7.7. A similar pH dependent inhibition was found when these compounds were examined for their effect on the hydrolysis of L-leucine-p-nitroanilide (LN) by serum aminopeptidases at pH between 6.2 and 7.7. Although the inhibitors were more effective against the hydrolysis of LN than BCN substrate, cGMP and its 8-bromo derivative were more active against the BCN hydrolysis. cAMP, 8-bromo-cAMP, dibutyryl (db)-cAMP, db-cGMP, AMP, ADP, ATP, GDP, GTP, aspirin, sodium salicylate, paracetamol, theophylline and isobutylmethylxanthine (IBMX) at comparable concentrations and within the same pH range had no effect on the hydrolysis of either substrate. It is concluded that in serum obtained during pregnancy, the hydrolysis of LN may largely be attributed to oxytocinase activity. Thus, inhibition of LN hydrolysis by prostaglandins and other substances may be regarded as inhibition of oxytocinase activity.

Leucine aminopeptidase (bovine lens). The relative binding of cobalt and zinc to leucine aminopeptidase and the effect of cobalt substitution on specific activity.

Prolonged incubation of zinc-zinc leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with 0.05 M CoCl2 and M KCl in 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees yields an active enzyme in which 2 g atoms of Co2+ per 54,000 dalton subunit have replaced the Zn2+. Incubation of cobalt-cobalt leucine aminopeptidase with various AnCl2 concentrations or zinc-zinc leucine aminopeptidase with various CoCl2 concentrations in M KCl and 0.2 M N-ethylmorpholine-HCl at pH 7.5 and 37 degrees demonstrates that Co2+ and Zn2+ compete reversibly for two independent binding sites per subunit for which the ratio of the association constants for Zn2+ and Co2+ (1KZn:1KCo = 1KZn/Co; 2KZn:2KCo = 2KZn/Co) are 115 and 15.9 for sites 1 and 2, respectively. The specific activities of the various species of enzyme with 2 mM L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl and 0.01 M NaHCO3 at pH 7.5 are estimated to be (in micromoles per min per mg) 0.043 for the zinc-zinc. 0.039 for the zinc-cobalt, 0.541 for the cobalt-zinc, and 0.536 for the cobalt-cobalt forms, which implies that activity is affected only when cobalt is substituted at site 1, the "activation site." The site, at which cobalt substitution has no effect on activity, is designated the "structural site." The value of Km for cobalt-cobalt leucine aminopeptidase with L-leucine p-nitroanilide as substrate in 0.2 M N-ethylmorpholine-HCl at pH 7.5 containing 0.01 M NaHCO3 at 30 degrees is 0.52 mM while Vmax is 0.90 mumol per min per mg. In the additional presence of 1 M KCl, Km is 0.19 mM while Vmax is 0.68 mumol per min per mg.