Functional human insulin-degrading enzyme can be expressed in bacteria

Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides. Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximately 8 mg per liter of culture). The recombinant IDEs, like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in SDS-polyacrylamide gels and as a approximately 200-kDa species in gel filtration. Further analysis by native PAGE indicates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stable at 4 degrees C for at least 1 month. Purified recombinant protein was used to raise specific polyclonal antibodies that can immunoprecipitate native mammalian IDE. Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian proteins.     

Insulin-degrading enzyme hydrolyzes ATP

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334-341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by (32)P release after hydrolysis of gamma[(32)P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 +/- 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 +/- 3.48 microM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.     

Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster

An insulin-degrading enzyme (IDE) from the cytoplasm of Drosophila Kc cells has been purified and characterized. The purified enzyme is a monomer with an s value of 7.2 S, an apparent Km for porcine insulin of 3 microM, and a specific activity of 3.3 nmol of porcine insulin degraded/(min.mg). N-Terminal sequence analysis of the gel-purified enzyme gave a single, serine-rich sequence. The Drosophila IDE shares a number of properties in common with its mammalian counterpart. The enzyme could be specifically affinity-labeled with [125I]insulin, has a molecular weight of 110K, and has a pI of 5.3. Although Drosophila Kc cells grow at room temperature, the optimal enzyme activity assay conditions parallel those of the mammalian IDE: 37 degrees C and a pH range of 7-8. The Drosophila IDE activity, like the mammalian enzymes, is inhibited by bacitracin and sulfhydryl-specific reagents. Similarly, the Drosophila IDE activity is insensitive to glutathione as well as protease inhibitors such as aprotinin and leupeptin. Insulin-like growth factor II, equine insulin, and porcine insulin compete for degradation of [125I]insulin at comparable concentrations (approximately 10(-6) M), whereas insulin-like growth factor I and the individual A and B chains of insulin are less effective. The high degree of evolutionary conservation between the Drosophila and mammalian IDE suggests an important role for this enzyme in the metabolism of insulin and also provides further evidence for the existence of a complete insulin-like system in invertebrate organisms such as Drosophila.     

Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme

Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid beta (Abeta). Tight interactions with substrates occur at an exosite located approximately 30 A away from the catalytic center that anchors the N-terminus of substrates to facilitate binding and subsequent cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic site and the exosite simultaneously. Here, we use kinins as a model system to address the kinetics and regulation of human IDE with short peptides. IDE specifically degrades bradykinin and kallidin at the Pro/Phe site. A 1.9 A crystal structure of bradykinin-bound IDE reveals the binding of bradykinin to the exosite and not to the catalytic site. In agreement with observed high K(m) values, this suggests low affinity of bradykinin for IDE. This structure also provides the molecular basis on how the binding of short peptides at the exosite could regulate substrate recognition. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S-nitrosylation and oxidation agents. Cysteine-directed modifications play a key role, since an IDE mutant devoid of all 13 cysteines is insensitive to the inhibition by S-nitrosoglutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing toward an extended hydrophobic pocket and is critical for the inactivation. Thiol-directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.     

Degradation of amylin by insulin-degrading enzyme

A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation.     

Proteolytic fragments of insulysin (IDE) retain substrate binding but lose allosteric regulation

Treatment of an N-terminal-containing His6-tagged insulysin (His6-IDE) with proteinase K led to the initial cleavage of the His tag and linker region. This was followed by C-terminal cleavages resulting in intermediate fragments of approximately 95 and approximately 76 kDa and finally a relatively stable approximately 56 kDa fragment. The approximately 76 and approximately 56 kDa fragments exhibited a low level of catalytic activity but retained the ability to bind the substrate with a similar affinity as the native enzyme. The kinetics of the reaction of the IDE approximately 76 and approximately 56 kDa proteolytic fragments with a synthetic fluorogenic substrate produced hyperbolic substrate versus velocity curves, rather than the sigmoidal curve obtained with His6-IDE. The approximately 76 and approximately 56 kDa IDE proteolytic fragments were active toward the physiological peptides beta-endorphin, insulin, and amyloid beta peptide 1-40. Although activity was reduced by a factor of approximately 103-104 with these substrates, the relative activity and the cleavage sites were unchanged. Both the approximately 76 and approximately 56 kDa fragments retained the regulatory cationic binding site that binds ATP. Thus, the two proteinase K cleavage fragments of IDE retain the substrate- and ATP-binding sites but have low catalytic activity and lose the allosteric kinetic behavior of IDE. These data suggest a role of the C-terminal region of IDE in allosteric regulation.     

Functional characterization of a maize ribosomal S6 protein kinase (ZmS6K), a plant ortholog of metazoan p70(S6K)

Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.     

Functional relevance of a novel SlyX motif in non-conventional secretion of insulin-degrading enzyme

Insulin-degrading enzyme (IDE) is a Zn(2+) metalloprotease with a characteristic inverted catalytic motif. IDE is ubiquitously expressed and degrades peptide substrates including insulin, endorphin, and the amyloid-β peptide. Although IDE is mainly expressed in the cytosol, it can also be found on the cell surface and in secreted form in extracellular fluids. As IDE lacks a characteristic signal sequence that targets the protein to the classical secretory pathway, release of the enzyme involves non-conventional mechanisms. However, functional domains of IDE involved in its secretion remain elusive. By bioinformatical, biochemical, and cell biological methods, we identified a novel amino acid motif ((853)EKPPHY(858)) close to the C terminus of IDE and characterized its function in the non-conventional secretion of the protein. Because of its close homology to an amino acid sequence found in bacterial proteins belonging to the SlyX family, we propose to call it the SlyX motif. Mutagenesis revealed that deletion of this motif strongly decreased the release of IDE, whereas deletion of a potential microbody-targeting signal at the extreme C terminus had little effect on secretion. The combined data indicate that the non-conventional secretion of IDE is regulated by the newly identified SlyX motif.     

Variations in Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Plants

Studies of ribulose-1,5-bisphosphate (RuBP) carboxylase from taxonomically diverse plants show that the enzyme from C(3) and crassulacean acid metabolism pathway species exhibits lower K(m)(CO(2)) values (12-25 micromolar) than does that from C(4) species (28-34 micromolar). RuBP carboxylase from aquatic angiosperms, an aquatic bryophyte, fresh water and marine algae has yielded consistently high K(m)(CO(2)) values (30-70 micromolar), similar in range to that of the enzyme from C(4) terrestrial plants. This variation in K(m)(CO(2)) is discussed in relation to the correlation between the existence of CO(2)-concentrating mechanisms for photosynthesis and the affinity of the enzyme for CO(2). The K(m)(RuBP) of the enzyme from various sources ranges from 10 to 136 micromolar; mean +/- sd = 36 +/- 20 micromolar. This variation in K(m)(RuBP) does not correlate with different photosynthetic pathways, but shows taxonomic patterns. Among the dicotyledons, the enzyme from crassinucellate species exhibits lower K(m)(RuBP) (18 +/- 4 micromolar) than does that from tenuinucellate species (25 +/- 7 micromolar). Among the Poaceae, RuBP carboxylase from Triticeae, chloridoids, andropogonoids, Microlaena, and Tetrarrhena has yielded lower K(m)(RuBP) values (29 +/- 11 micromolar) than has that from other members of the grass family (46 +/- 10 micromolar).     

Biochemical properties of a putative signal peptide peptidase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppA(Tk)) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppA(Tk), comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (deltaN54SppA(Tk)) was found to be stable against autoproteolysis and was examined further. DeltaN54SppA(Tk) exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. DeltaN54SppA(Tk) displayed a K(m) of 240 +/- 18 microM and a V(max) of 27.8 +/- 0.7 micromol min(-1) mg(-1) towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80 degrees C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, deltaN54SppA(Tk) was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of deltaN54SppA(Tk) was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.     

Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-α, and Amylin by Human Insulin-Degrading Enzyme

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-β, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-α (TGF-α) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-α, amylin, reduced amylin, and amyloid-β by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-α at 2.3 Å and IDE-amylin at 2.9 Å. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.     

Peptidyl dipeptidases (Ance and Acer) of Drosophila melanogaster: major differences in the substrate specificity of two homologs of human angiotensin I-converting enzyme

Drosophila melanogaster angiotensin converting enzyme (Ance) and angiotensin converting enzyme related (Acer) are single domain homologs of mammalian peptidyl dipeptidase A (angiotensin I-converting enzyme) whose physiological substrates have not as yet been identified. We have investigated the in vitro substrate specificities of the two peptidases towards a variety of insect and mammalian peptides. Ance was generally much better than Acer at hydrolyzing peptides of 5-13 amino acids in length. Only two of the peptides, [Leu(5)]enkephalinamide and leucokinin-I were cleaved faster by Acer. Increasing NaCl concentration had opposite affects on the cleavage of [Leu(5)]enkephalin and [Leu(5)]enkephalinamide by Acer, decreasing the activity towards [Leu(5)]enkephalin but increasing the activity towards [Leu(5)]enkephalinamide. Of the insect peptides tested, the tachykinin-related peptide, Lom TK-1, proved to be the best substrate for Ance with a k(cat)/K(m) ratio of 0.122s(-1) microM(-1). However, in comparison, the D. melanogaster tachykinins, DTK-1, DTK-2, DTK-3 and DTK-4 were poor Ance substrates. DTK-5 was the best substrate of this family, but the apparent high K(m) for hydrolysis by Ance suggested that this peptide would not be a natural Ance substrate. This low affinity for DTK-5 is the likely reason why the peptide was not rapidly degraded in D. melanogaster hemolymph, where Ance was shown to be a major peptide-degrading activity.     

Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase

Phytases are enzymes that catalyze liberation of inorganic phosphates from phytate, the major organic phosphorus in soil. Tobacco (Nicotiana tabacum) responds to phosphorus starvation with an increase in extracellular phytase activity. By a three-step purification scheme, a phosphatase with phytase activity was purified 486-fold from tobacco root exudates to a specific activity of 6,028 nkat mg(-1) and an overall yield of 3%. SDS-PAGE revealed a single polypeptide of 64 kDa, thus indicating apparent homogeneity of the final enzyme preparation. Gel filtration chromatography suggested that the enzyme was a ca. 56 kDa monomeric protein. De novo sequencing by tandem mass spectrometry resulted in a tryptic peptide sequence that shares high homology with several plant purple acid phosphatases. The identity of the enzyme was further confirmed by molybdate-inhibition assay and cDNA cloning. The purified enzyme exhibited pH and temperature optima at 5.0-5.5 and 45 degrees C, respectively, and were found to have high affinities for both p-nitrophenyl phosphate (pNPP; K(m)=13.9 microM) and phytate (K(m)=14.7 microM), but a higher kcat for pNPP (2,056 s(-1)) than phytate (908 s(-1)). Although a broad specificity of the enzyme was observed for a range of physiological substrates in soil, maximum activity was achieved using mononucleotides as substrates. We conclude that the phytase activity in tobacco root exudates is exhibited by a purple acid phosphatase and its catalytic properties are pertinent to its role in mobilizing organic P in soil.     

Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions

The active site glutamate (Glu(111)) and the active site histidine (His(112)) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient > 2), to a hyperbolic response. With 2-aminobenzoyl-GGFLRKHGQ-N-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. The His(112) mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. The glutamate mutants displayed an altered cleavage profile for the peptide beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme.     

Mixed dimers of insulin-degrading enzyme reveal a cis activation mechanism

Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity. The mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. This effect shows that substrate or small peptide activation occurs through a cis effect. A mixed dimer composed of one wild-type subunit and the other subunit containing a mutation that neither permits substrate binding nor catalysis (H112Q) exhibits the same turnover number per active subunit as wild-type IDE. In contrast, a mixed dimer in which one subunit contains the wild-type sequence and the other contains a mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. This indicates a negative trans effect of substrate binding at the active site. On the other hand, activation in trans is observed with extended substrates that occupy both the active and distal sites. Comparison of the binding of an amyloid β peptide analog to wild-type IDE and to the Y609F mutant showed no difference in affinity, indicating that Y609 does not play a significant role in substrate binding at the distal site.     

Substrate activation of insulin-degrading enzyme (insulysin). A potential target for drug development

The rate of the insulin-degrading enzyme (IDE)-catalyzed hydrolysis of the fluorogenic substrate 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl is increased 2-7-fold by other peptide substrates but not by peptide non-substrates. This increased rate is attributed to a decrease in Km with little effect on Vmax. An approximately 2.5-fold increase in the rate of amyloid beta peptide hydrolysis is produced by dynorphin B-9. However, with insulin as substrate, dynorphin B-9 is inhibitory. Immunoprecipitation of differentially tagged IDE and gel filtration analysis were used to show that IDE exists as a mixture of dimers and tetramers. The equilibrium between dimer and tetramer is concentration-dependent, with the dimer the more active form. Bradykinin shifted the equilibrium toward dimer. Activation of substrate hydrolysis is not seen with a mixed dimer of IDE containing one active subunit and one subunit that is catalytically inactive and deficient in substrate binding. On the other hand, a mixed dimer containing one active subunit and one subunit that is catalytically inactive but binds substrate with normal affinity is activated by peptides. These findings suggest that peptides bind to one subunit of IDE and induce a conformational change that shifts the equilibrium to the more active dimer as well as activates the adjacent subunit. The selective activation of IDE toward amyloid beta peptide relative to insulin suggests the potential for development of compounds that increase IDE activity toward amyloid beta peptide as a therapeutic intervention for the treatment of Alzheimer's disease.     

Digestive proteinases of yellow mealworm (<Emphasis Type="Italic">Tenebrio molitor</Emphasis>) larvae: Purification and characterization of a trypsin-like proteinase

A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55 degrees C, pH optimum at 8.5, and K(m) value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.     

Liver prenylated methylated protein methyl esterase is the same enzyme as Sus scrofa carboxylesterase

The C-terminal --COOH of prenylated proteins is methylated to --COOCH3. The --COOCH3 ester forms are hydrolyzed by prenylated methylated protein methyl esterase (PMPMEase) to the original acid forms. This is the only reversible step of the prenylation pathway. PMPMEase has not been purified and identified and is therefore understudied. Using a prenylated-L-cysteine methyl ester as substrate, PMPMEase was purified to apparent homogeneity from porcine liver supernatant. SDS-PAGE analysis revealed an apparent mass of 57 kDa. Proteomics analyses identified 17 peptides (242 amino acids). A Mascot database search revealed these as portions of the Sus scrofa carboxylesterase, a 62-kDa serine hydrolase with the C-terminal HAEL endoplasmic reticulum-retention signal. It is at least 71% identical to such mammalian carboxylesterases as human carboxylesterase 1 with affinities toward hydrophobic substrates and known to activate prodrugs, metabolize active drugs, as well as detoxify various substances such as cocaine and food-derived esters. The purified enzyme hydrolyzed benzoyl-Gly-farnesyl-L-cysteine methyl ester and hydrocinamoyl farnesyl-L-cysteine methyl ester with Michaelis-Menten constant (K(m)) values of 33 +/- 4 and 25 +/- 4 microM and V(max) values of 4.51 +/- 0.28 and 6.80 +/- 0.51 nmol/min/mg of protein, respectively. It was inhibited by organophosphates, chloromethyl ketones, ebelactone A and B, and phenylmethylsulfonyl fluoride.     

Cloning, expression, and characterization of tomato (Lycopersicon esculentum) aminopeptidase P.

A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase and was purified from bacterial extracts. LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K(m) (15.2 +/- 2.4 microm) and K(m)/k(cat) (0.94 +/- 0.11 mm(-1) x s(-1)) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate. LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl(2) and to some extent also by Mg(2+), Ca(2+), and Co(2+), whereas other divalent metal ions (Cu(2+), Zn(2+)) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P.