Purification and partial characterization of an intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114.

An intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100. The enzyme had a molecular weight of 89,000. It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35 degrees C with Km = 1.80 mmol/l; above 55 degrees C the enzyme activity declined rapidly. The aminopeptidase was capable of degrading substrates by hydrolysis of the N-terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity. The enzyme was strongly inactivated by EDTA. Serine and sulphydryl group reagents had no effect on enzyme activity.     

The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction

Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was approximately 100-fold less sensitive toward puromycin (IC50, 135 mum) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.     

Hydrosoluble fluorogenic substrates for plasmin.

New hydrosoluble fluorogenic substrates for plasmin gluconoylpeptidyl-3-amido-9-ethylcarbazole were synthesized. The substitution of the N-terminal end of the peptides by a gluconoyl group prevents the substrates from aminopeptidase degradation and highly increases their hydrosolubility. The substitution of the peptide C-terminal end by a 3-amino-9-ethylcarbazole group leads to substrates suitable for direct fluorometric assay of plasmin present in cell supernatants or in cell lysates. On the basis of the kinetic parameters of the substrate hydrolysis by plasmin, it was found that D amino acids in the P2 position decrease systematically the kinetic constants of the substrates. The L configuration of the P2 amino acid appears therefore as essential in optimum substrates for plasmin.     

A non-specific aminopeptidase from Aspergillus

A fermentation broth supernatant of the Aspergillus oryzae strain ATCC20386 contains aminopeptidase activity that releases a wide variety of amino acids from natural peptides. The supernatant was fractionated by anion exchange chromatography. Based on the primary amino acid sequence data obtained from proteins in certain fractions, polymerase chain reaction (PCR) primers were made and a PCR product was generated. This PCR product was used to screen an A. oryzae cDNA library from which the full length gene was then obtained. Fusarium venenatum and A. oryzae were used as hosts for gene expression. Transformed strains of both F. venenatum and A. oryzae over-expressed an active aminopeptidase (E.C. 3.4.11), named aminopeptidase II. The recombinant enzyme from both fungal hosts appeared as smears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After deglycosylation of the N-linked sugars, both samples were a sharp band at approximately 56 kDa and had identical N-terminal amino acid sequences. Aminopeptidase II is a metalloenzyme with, presumably, Zn in the active site. Using various natural peptides and para-nitroanilides (pNAs) of amino acids as substrates, the aminopeptidase was found to be non-specific. Only X-Pro bonds demonstrated resistance to hydrolysis catalyzed by this aminopeptidase. The optimal enzyme activity was observed at pH 9.5 and 55 degrees C. Among amino acid pNAs, Leu-pNA appears to have the highest value of bimolecular constant of 40 min(-1) mM(-1) (k(cat) = 230 min(-1); K(m) = 5.8 mM) at pH 7.5 and 21 degrees C. Among Xaa-Ala-Pro-Tyr-Lys-amide pentapeptides, the velocity of catalytic hydrolysis at pH 7.5 and 21 degrees C was in a decreasing order: Pro, Ala, Leu, Gly and Glu.     

The initiation of haemoglobin synthesis in rabbit reticulocytes

1. The incorporation of labelled valine by rabbit reticulocytes into the N-terminal position of nascent haemoglobin was investigated by deaminating the nascent peptides with nitrous acid and isolating labelled alpha-hydroxyisovaleric acid and valine after acid hydrolysis. 2. The amount of radioactivity in alpha-hydroxyisovaleric acid relative to that in valine indicated the presence of 12.3% N-terminal valine having a free amino group. This high value suggests that most if not all nascent peptides contain valine in the N-terminal position. 3. Cell-free preparations containing reticulocyte ribosomes and pH5 enzymes incorporated alpha-hydroxy-[(14)C]isovaleryl-tRNA (where tRNA refers to transfer RNA), which was obtained by deamination of [(14)C]valyl-tRNA from yeast or liver with nitrous acid, into both soluble and nascent protein. 4. When the soluble protein was chromatographed on CM-cellulose, radioactivity was found to be associated with both the alpha-and beta-globin chains. 5. The kinetics of hydrolysis of [(14)C]valine, was also investigated. Most of the material was hydrolysed rapidly at pH10, but a minor component that was relatively stable appeared to be present to the extent of about 10% of the total valyl-tRNA. Valine was, however, the only hydrolysis product detected by paper chromatography. 6. It is concluded that chain initiation in haemoglobin synthesis involves valine as the N-terminal amino acid and that the amino group of nascent protein is probably not substituted.     

Purification of and kinetic studies on a narrow specificity synaptosomal membrane pyroglutamate aminopeptidase from guinea-pig brain

A pyroglutamate aminopeptidase activity, distinct from that of cytoplasm, was released from a synaptosomal membrane preparation of guinea-pig brain by papain treatment. This activity was further purified 3560-fold relative to the homogenate with a yield of 17% by a procedure involving gel filtration chromatography, calcium phosphate cellulose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The purified synaptosomal pyroglutamate aminopeptidase hydrolysed only thyroliberin, acid-thyroliberin, the luliberin N-terminal tripeptide (Glp-His-Trp) and, only slightly, Glp-His-Gly. No hydrolysis was observed with dipeptides containing N-terminal pyroglutamic acid (Glp) or with pyroglutamyl peptides containing more than three amino acids. A Km value of 40 microM was recorded when thyroliberin was used as substrate; however, luliberin was found to inhibit the hydrolysis of thyroliberin competitively with a Ki value of 20 microM.     

Sequential hydrolysis of proline-containing peptides with immobilized aminopeptidases

Proline-containing polypeptides are shown to be sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains, but does not cleave the N-terminal secondary peptide bonds involving a prolyl nitrogen. Aminopeptidase P (EC 3.4.11.9) cleaves exclusively such secondary bonds. The two enzymes were immobilized by coupling them covalently to porous amino glass beads. Highly stable preparations were obtained with unchanged pH optimum and thermal stability. The applicability of clostridial aminopeptidase to sequence determination was demonstrated by the time-dependent hydrolysis of enkephalin and Substance P octapeptide. Sequential hydrolysis with the two immobilized enzymes was demonstrated with the proline-containing (Pro-Gly-Pro)10, [Asn1, Val5]angiotensin II, bradykinin, Substance P and tuftsin. Absence of endopeptidase activities was demonstrated by resistance of cytochrome c to hydrolysis and by the ordered release of amino acids during the sequential degradation by immobilized clostridial aminopeptidase and aminopeptidase P.     

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.     

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.     

Novel chromogenic aminopeptidase substrates for the detection and identification of clinically important microorganisms

A series of amino acid derivatives 8–10, 42 and 43 have been prepared as chromogenic enzyme substrates in order to detect aminopeptidase activity in clinically important Gram-negative and Gram-positive bacteria. Enzymatic hydrolysis liberates the amino acid moiety and either a 4-aminophenol or a 4-dialkylaminoaniline derivative which undergoes oxidative coupling with 1-naphthol or a substituted 1-naphthol giving an indophenol dye. Substrates and 1-naphthols were incorporated into an agar-based culture medium and this allowed growth of intensely coloured bacterial colonies based on hydrolysis by specific enzymes. Red/pink coloured colonies were produced by the substrates 8–10 and blue coloured colonies were formed by the substrates 42 and 43. The l-alanyl aminopeptidase substrates 8 targeted l-alanyl aminopeptidase activity and gave coloured colonies with a range of Gram-negative bacteria. Substrates 9 targeted β-alanyl aminopeptidase activity and generated coloured colonies with selected Gram-negative species including Pseudomonas aeruginosa. Three substrates for l-pyroglutamyl acid aminopeptidase (10a, 10c and 43) were hydrolysed by enterococci and Streptococcus pyogenes to generate coloured colonies. Two yeasts were also included in the study, but they did not produce coloured colonies with any of the substrates examined.     

Bitter Peptide Isolated from Milk Cultures of Streptococcus cremoris

Certain cultures of Streptococcus cremoris produced a bitter taste that occurred in the whey portion of milk cultures. Whey from a culture which produced bitterness was fractionated on Sephadex. The fraction in which the bitter taste was concentrated was chromatographed successively on paper with butanol-acetic acid-water (5:1:4), and then butanol-2-butanone-water (2:2:1). In each instance, the bitter component was in the most rapidly moving band that gave a positive ninhydrin test. The bitterness was observed to be caused by a peptide containing the following numbers of each amino acid: arginine, 1; glutamic acid, 2; glycine, 2; isoleucine, 2; leucine, 2; phenylalanine, 1; proline, 5; and valine, 4. N-terminal amino acids could be detected by coupling with 2,4-dinitrofluorobenzene or phenylisothiocyanate, or by hydrolysis with leucine aminopeptidase. When treated with carboxypeptidase, only leucine and valine appeared at the C-terminal end, and these were detected simultaneously.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from beta-lipotropin60-65

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane-bound aminopeptidase activity which cleaved arginine from beta-LPH60-65 (Arg-Tyr-Gly-Gly-Phe-Met) and Arg-MCA. Neither methionine enkephalin (Tyr-Gly-Gly-Phe-Met) nor Substance P, which has an N-terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B-like or chymotrypsin-like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+ and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p-chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.     

Isolation, purification and investigation of physico-chemical properties and specificity of Leu-Gly-Gly-amino peptidase]

A highly purified (237-fold) preparation of extracellular Leu-Gly-Gly aminopeptidase was isolated from the 716 strain of mould Aspergillis flavus. The enzyme was found electrophoretically and enzymatically homogeneous, using Leu-beta-naphthylimide as substrate. The pH optimum is 8.60; the temperature optimum is about 50 degrees C. The enzyme was inhibited by EDTA and completely reactivated by Co2+ ions; Ca2+ and Mn2+ ions considerably restored the enzyme activity. The enzyme showed the optimal activity during the cleavage of substrates, containing N-terminal leucine. Mild hydrolysis of leucine-free tripeptides and dipeptides with N-terminal glycine and alanine was observed. The enzyme was found to be stereospecific in some respects. Peptides with a blocked terminal NH2-group are not hydrolyzed by the enzyme.     

RNA determinants for translational editing. Mischarging a minihelix substrate by a tRNA synthetase

The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TPsiC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system.     

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.     

Degradation of thymic humoral factor gamma2 by human plasma: involvement of angiotensin converting enzyme

The degradation of thymic humoral factor-gamma2 (THF-gamma2), an immunoregulatory octapeptide important for T-lymphocyte regulation, by enzymes present in human plasma, was investigated. THF-gamma2 was metabolized through two steps that involved the detaching of N-terminal amino acid leucine followed by hydrolysis of the Lys(6)-Phe(7) bond. The THF-gamma2 cleavages were sensitive to aminopeptidase and metalloproteinase inhibitors. The degradation was completely blocked by amastatin and specific inhibitors of angiotensin converting enzyme (ACE). The cleavages occurred independently, with two different kinetics, faster for the N-terminal hydrolysis than for that of the Lys(6)-Phe(7) bond. Purified human plasma ACE was used to characterize the hydrolysis of Lys(6)-Phe(7) bond. The K(m) and K(cat) values for THF-gamma2 hydrolysis were 0.273 mM and 107 s(-1), respectively. The optimum of chloride concentration was 300 mM, while that of pH was 7.6. The presence of ACE in circulating mononuclear cells raises the possibility that it may play a role in modulating the THF-gamma2 activity.     

Thermostable aminopeptidase from Pyrococcus horikoshii

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.     

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.     

Characterization of two new aminopeptidases in Escherichia coli

Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity.