Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesis.

In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards L-methionine, L-norleucine and L-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used L-aspartate and L-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher K(m ) values for glutamate/2-oxoglutarate at pH 7.0, but a similar k(cat)/K(m) value and lower K(m) at pH 8.0, and a nearly 22-fold lower S(0.5) (substrate concentration giving half-saturation under conditions where Michaelis-Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100-1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially L-norleucine and L-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate k(cat) and K(m) separately, but for reductive amination the additional mutations have no significant effect on k(cat) at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in K(m). At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine approximately 2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants.     

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.     

Overexpression and characterization of a carboxypeptidase from the hyperthermophilic archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from Pyrococcus furiosus, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in Escherichia coli. Molecular masses assessed by SDS-PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70-80 degrees C (k(cat)/K(m)=8.3 mM(-1) s(-1)). In comparison with that of P. furiosus CP (k(cat)/K(m)=667 mM(-1) s(-1)), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine N-acetyl-renin substrate by TNA1_CP.     

绮丽刺毛霉的一种新型甘氨酸氨肽酶的研究

研究了产自于绮丽刺毛霉（Actinomucor elegans）的一种甘氨酸氨肽酶。分子筛层析表明该酶的天然分子的分子量为320kD,SDSPAGE分析表明蛋白质的亚基分子量为565kD。该酶水解含有甘氨酸残基的底物（如glycinenaphthylamine）的效率要较其它氨基酸残基高得多。该酶的最佳反应温度为30℃,最佳pH为8.0。酶的Km和Kcat值分别为0.24mmol/L与1008 s^-1。1.0mmol/L Zn²⁺,Cu²⁺和Cd²⁺可完全抑制该酶的活性。作用于酶巯基的化学物质对酶活性都有抑制作用。根据络合剂反应的实验结果表明该酶是一种含有金属的酶。当与蛋白酶共同作用时该酶除了甘氨酸外还能提高脯氨酸、精氨酸及谷氨酸的水解率。     

Identification of a puromycin-sensitive aminopeptidase from zebrafish (Danio rerio)

An aminopeptidase from zebrafish (Danio rerio) was purified 1247-fold to homogeneity with 35.4% recovery by column chromatography successively on DEAE-sephacel, hydroxyapatite, and phenyl-sepharose. The molecular mass of the enzyme was estimated at 98 kDa by SDS-PAGE and gel filtration. Optimum temperature and pH of the enzyme were 45°C and 7.5, respectively. The enzyme preferentially hydrolyzed substrate Leu-MCA with k(cat)/K(m) of 4.2×10(6)M(-1)s(-1) and an activation energy of 68.9 kJ M(-1) [corrected], respectively. It was specifically inhibited by bestatin, puromycin and metal-chelating agents, and Zn(2+) seemed to be its metal cofactor(s). Some l-amino acids significantly inhibited its activity, and l-cysteine was a non-competitive inhibitor with a K(i) of 0.27 mM. According to the peptide mass fingerprint analysis, the enzyme was highly matched with the predicted D. rerio aminopeptidase puromycin sensitive (gi: 255683530) (EC 3.4.11.14), suggesting that the present enzyme is a puromycin-sensitive aminopeptidase of zebrafish.     

A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110kDa has an optimal pH of 7.0 and a pI of 5.6. It splits beta-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl beta-naphthylamide (Leu betaNA) is the best substrate with the highest hydrolytic coefficiency followed by Met betaNA=Arg betaNA=Lys betaNA>Ala betaNA>Tyr betaNA>Phe betaNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K(m) 82microM and a k(cat) of 1.08s(-1), and Met-enkephalin with a K(m) of 106microM and a k(cat) of 2.6s(-1). The puromycin-sensitive enzyme is most susceptible to amastatin with an IC(50) of 0.05microM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.     

Designing of substrates and inhibitors of bovine alpha-chymotrypsin with synthetic phenylalanine analogues in position P(1)

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.     

Peptidase specificity characterization of C- and N-terminal catalytic sites of angiotensin I-converting enzyme

Quenched fluorescence peptides were used to investigate the substrate specificity requirements for recombinant wild-type angiotensin I-converting enzyme (ACE) and two full-length mutants bearing a single functional active site (N- or C-domain). We assayed two series of bradykinin-related peptides flanked by o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp), namely, Abz-GFSPFXQ-EDDnp and Abz-GFSPFRX-EDDnp (X = natural amino acids), in which the fluorescence appeared when Abz/EDDnp are separated by substrate hydrolysis. Abz-GFSPFFQ-EDDnp was preferentially hydrolyzed by the C-domain while Abz-GFSPFQQ-EDDnp exhibits higher N-domain specificity. Internally quenched fluorescent analogues of N-acetyl-SDKP-OH were also synthesized and assayed. Abz-SDK(Dnp)P-OH, in which Abz and Dnp (2,4-dinitrophenyl) are the fluorescent donor-acceptor pair, was cleaved at the D-K(Dnp) bond with high specificity by the ACE N-domain (k(cat)/K(m) = 1.1 microM(-)(1) s(-)(1)) being practically resistant to hydrolysis by the C-domain. The importance of hydroxyl-containing amino acids at the P(2) position for N-domain specificity was shown by performing the kinetics of hydrolysis of Abz-TDK(Dnp)P-OH and Abz-YDK(Dnp)P-OH. The peptides Abz-YRK(Dnp)P-OH and Abz-FRK(Dnp)P-OH which were hydrolyzed by wild-type ACE with K(m) values of 5.1 and 4.0 microM and k(cat) values of 246 and 210 s(-)(1), respectively, have been shown to be excellent substrates for ACE. The differentiation of the catalytic specificity of the C- and N-domains of ACE seems to depend on very subtle variations on substrate-specific amino acids. The presence of a free C-terminal carboxyl group or an aromatic moiety at the same substrate position determines specific interactions with the ACE active site which is regulated by chloride and seems to distinguish the activities of both domains.     

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.     

Engineering the substrate specificity of Alcaligenes D-aminoacylase useful for the production of D-amino acids by optical resolution

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of D-amino acid from the racemic mixture of N-acyl-DL-amino acids. However, its substrate specificity is biased toward certain N-acyl-D-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other D-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (k(cat)/K(m)) of mutant F191W toward N-acetyl-D-Trp and N-acetyl-D-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (k(cat)/K(m)) of mutant L298A toward N-acetyl-D-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of D-Trp which is an important building block of some therapeutic drugs.     

Mutation of active site residues of the puromycin-sensitive aminopeptidase: conversion of the enzyme into a catalytically inactive binding protein

The active site glutamate, Glu 309, of the puromycin-sensitive aminopeptidase was mutated to glutamine, alanine, and valine. These mutants were characterized with amino acid beta-naphthylamides as substrates and dynorphin A(1-9) as an alternate substrate inhibitor. Conversion of glutamate 309 to glutamine resulted in a 5000- to 15,000-fold reduction in catalytic activity. Conversion of this residue to alanine caused a 25,000- to 100,000-fold decrease in activity, while the glutamate to valine mutation was the most dramatic, reducing catalytic activity 300,000- to 500,000-fold. In contrast to the dramatic effect on catalysis, all three mutations produced relatively small (1.5- to 4-fold) effects on substrate binding affinity. Mutation of a conserved tyrosine, Y394, to phenylalanine resulted in a 1000-fold decrease in k(cat), with little effect on binding. Direct binding of a physiological peptide, dynorphin A(1-9), to the E309V mutant was demonstrated by gel filtration chromatography. Taken together, these data provide a quantitative assessment of the effect of mutating the catalytic glutamate, show that mutation of this residue converts the enzyme into an inactive binding protein, and constitute evidence that this residue acts a general acid/base catalyst. The effect of mutating tyrosine 394 is consistent with involvement of this residue in transition state stabilization.     

Substrate specificity of the oncoprotein v-Fps: site-specific mutagenesis of the putative P+1 pocket

Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J. 16, 5572-5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predicted to interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an important recognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E). The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine, or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrate concentrations (i.e., k(cat)/K(m) conditions) indicates that substrate specificity is altered by the electrostatic environment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), no charge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptide over the lysine peptide by a 200:1, 9:1, or 1:1 margin. While k(cat)/K(m) for the glutamate peptide is 50-fold higher for wild type compared to R1130E, k(cat)/K(m) for the lysine peptide is 3-fold higher for R1130E compared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual steps in the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds the glutamate peptide 3-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide. For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed. Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamate peptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rate by 4-30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimum catalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substrate specificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relative changes in peptide binding affinity.     

Purification and properties of a novel glycine amino peptidase from Actinomucor elegans and its potential application

AIMS: To study the properties and show the potential application of a glycine aminopeptidase from Actinomucor elegans. METHODS AND RESULTS: The enzyme was estimated to have molecular mass of 320 kDa by gel filtration and the subunit size of 56.5 kDa by SDS-PAGE. It hydrolysed glycine from substrate more efficiently than other amino acids. The optimal temperature for this enzyme was 40 degrees C and at pH 8.0 it showed its highest activity. The Km and Kcat of the enzyme for glycine-beta-naphthylamine was 0.24 mm and 100.8 s(-1), respectively. Zinc, copper, cadmium and o-phenanthrolin suppressed almost all enzyme activities at the concentration of 1.0 mm. In the process of hydrolysing proteins, it could improve the protease activity considerably. CONCLUSIONS: It was a hexamer metalloenzyme which was specific for the substrates with glycinse residue at the N-terminal and some metal cations were needed to maintain its activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the properties of a novel aminopeptidase and shows its potential application in the process of the food industry.     

On the catalytic role of the conserved active site residue His466 of choline oxidase

The oxidation of alcohols to aldehydes is catalyzed by a number of flavin-dependent enzymes, which have been grouped in the glucose-methanol-choline oxidoreductase enzyme superfamily. These enzymes exhibit little sequence similarity in their substrates binding domains, but share a highly conserved catalytic site, suggesting a similar activation mechanism for the oxidation of their substrates. In this study, the fully conserved histidine residue at position 466 of choline oxidase was replaced with an alanine residue by site-directed mutagenesis and the biochemical, spectroscopic, and mechanistic properties of the resulting CHO-H466A mutant enzyme were characterized. CHO-H466A showed k(cat) and k(cat)/K(m) values with choline as substrate that were 60- and 1000-fold lower than the values for the wild-type enzyme, while the k(cat)/K(m) value for oxygen was unaffected, suggesting the involvement of His(466) in the oxidation of the alcohol substrate but not in the reduction of oxygen. Replacement of His(466) with alanine significantly affected the microenvironment of the flavin, as indicated by the altered behavior of CHO-H466A with sulfite and dithionite. In agreement with this conclusion, a midpoint reduction potential of +106 mV for the two-electron transfer in the catalytically competent enzyme-product complex was determined at pH 7 for CHO-H466A, which was approximately 25 mV more negative than that of the wild-type enzyme. Enzymatic activity in CHO-H466A could be partially rescued with exogenous imidazolium, but not imidazole, consistent with the protonated form of histidine exerting a catalytic role. pH profiles for glycine betaine inhibition, the deprotonation of the N(3)-flavin locus, and the k(cat)/K(m) value for choline all showed a significant shift upward in their pK(a) values, consistent with a change in the polarity of the active site. Finally, kinetic isotope effects with isotopically labeled substrate and solvent indicated that the histidine to alanine substitution affected the timing of substrate OH and CH bond cleavages, consistent with removal of the hydroxyl proton being concerted with hydride transfer in the mutant enzyme. All taken together, the results presented in this study suggest that in choline oxidase, His(466) modulates the electrophilicity of the enzyme-bound flavin and the polarity of the active site, and contributes to the stabilization of the transition state for the oxidation of choline to betaine aldehyde.     

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.     

Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases.

Aminopeptidase A (EC 3.4.11.7, APA) is a 160 kDa membrane-bound zinc enzyme that contains the HEXXH consensus sequence found in members of the zinc metalloprotease family, the zincins. In addition, the monozinc aminopeptidases are characterized by another conserved motif, GXMEN, the glutamate residue of which has been shown to be implicated in the exopeptidase specificity of aminopeptidase A [Vazeux G. (1998) Biochem. J. 334, 407-413]. In carboxypeptidase A (EC 3.4.17.1, CPA), the exopeptidase specificity is conferred by an arginine residue (Arg-145) and an asparagine residue (Asn-144). Thus, we hypothesized that Asn-353 of the GXMEN motif in APA plays a similar role to Asn-144 in CPA and contributes to the exopeptidase specificity of APA. We investigated the functional role of Asn-353 in APA by substituting this residue with a glutamine (Gln-353), an alanine (Ala-353) or an aspartate (Asp-353) residue by site-directed mutagenesis. Expression of wild-type and mutated APAs revealed that Gln-353 and Ala-353 are similarly routed and glycosylated to the wild-type APA, whereas Asp-353 is trapped intracellularly and partially glycosylated. Kinetic studies, using alpha-L-glutamyl-beta-naphthylamide (GluNA) as a substrate showed that the K(m) values of the mutants Gln-353 and Ala-353 were increased 11- and 8-fold, respectively, whereas the k(cat) values were decreased (2-fold) resulting in a 24- and 14-fold reduction in cleavage efficiency. When alpha-L-aspartyl-beta-naphthylamide or angiotensin II were used as substrates, the mutations had a greater effect on k(cat), leading to a similar decrease in cleavage efficiencies as that observed with GluNA. We then measured the inhibitory potencies of several classes of inhibitors, glutamate thiol, glutamine thiol and two isomers (L- or D-) of glutamate phosphonate to explore the functional role of Asn-353. The data indicate that Asn-353 is critical for the integrity and catalytic activity of APA. This residue is involved in substrate binding via interactions with the free N-terminal part and with the P1 carboxylate side chain of the substrate. In conclusion, Asn-353 of the GXMEN motif, together with Glu-352, contributes to the exopeptidase specificity of APA and plays an equivalent role to Asn-144 in CPA.     

Orf135 from Escherichia coli Is a Nudix hydrolase specific for CTP, dCTP, and 5-methyl-dCTP

Orf135 from Escherichia coli is a new member of the Nudix (nucleoside diphosphate linked to some other moiety, x) hydrolase family of enzymes with substrate specificity for CTP, dCTP, and 5-methyl-dCTP. The gene has been cloned for overexpression, and the protein has been overproduced, purified, and characterized. Orf135 is most active on 5-methyl-dCTP (k(cat)/K(m) = 301,000 M(-1) s(-1)), followed by CTP (k(cat)/K(m) = 47,000 M(-1) s(-1)) and dCTP (k(cat)/K(m) = 18,000 M(-1) s(-1)). Unlike other nucleoside triphosphate pyrophophohydrolases of the Nudix hydrolase family discovered thus far, Orf135 is highly specific for pyrimidine (deoxy)nucleoside triphosphates. Like other Nudix hydrolases, the enzyme cleaves its substrates to produce a nucleoside monophosphate and inorganic pyrophosphate, has an alkaline pH optimum, and requires a divalent metal cation for catalysis, with magnesium yielding optimal activity. Because of the nature of its substrate specificity, Orf135 may play a role in pyrimidine biosynthesis, lipid biosynthesis, and in controlling levels of 5-methyl-dCTP in the cell.     

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.