Sulphonamide-based bombesin prodrug analogues for glutathione transferase, useful in targeted cancer chemotherapy.

Glutathione transferases (GSTs) are enzymes involved in cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of toxic compounds and xenobiotics, including certain chemotherapeutic drugs. The encountered chemotherapeutic resistant of tumour cells, thus, has been associated with the increase of total GST expression. GSTs, in addition to GSH-conjugating activity, exhibit sulphonamidase activity, catalyzing the GSH-mediated hydrolysis of sulphonamide bonds. Such reactions are of interest as potential tumour-directed prodrug activation strategies. In the present work we report the design and synthesis of novel chimaeric sulphonamide derivatives of bombesin, able to be activated by the model human isoenzyme GSTA1-1 (hGSTA1-1). These derivatives bear a peptidyl-moiety (analogues of bombesin peptide: R-[Lue(13)]-bombesin, R-[Phe(13)]-bombesin and R-[Ser(3),Arg(10),Phe(13)]-bombesin, where R=C(6)H(5)SO(2)NH-) as molecular recognition element for targeting the drug selectively to tumour cells. The released S-alkyl-glutathione, after hGSTA1-1-mediated cleavage of the sulphonamide bond, provides an inhibitor of varied strength against GSTs from different sources. These prodrugs are envisaged as a plausible means to sensitize drug-resistant tumours that overexpress GSTs.

Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes One-step purification of Pfu DNA polymerase.

The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (K(D)) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.

Interaction of L-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography.

Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of L-glutamate to alpha-ketoglutarate. Its kinetic constants for L-glutamate were measured equal to 2 mM for Km and 85.8 s(-1) for kcat. BLAST search and amino acid sequence alignments revealed low homology to other L-amino acid oxidases (18-38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic alpha-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting L-glutamate oxidase activity. All but Cibacron Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye-ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a Ki of 10.5 microM, indicating that the dye-enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg(1/2)ml(1/2)/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for L-glutamate oxidase.

Designed chimaeric galactosyl-mimodye ligands for the purification of Pseudomonas fluorescens beta-galactose dehydrogenase.

Two chimaeric galactosyl-mimodye ligands were designed and applied to the purification of Pseudomonas fluorescens galactose dehydrogenase (GaDH). The chimaeric affinity ligands comprised a triazine ring on which were anchored: (i) an anthraquinone moiety that pseudomimics the adenine part of NAD+, (ii) a galactosyl-mimetic moiety (D-galactosamine for ligand BM1 or shikimate for ligand BM2), bearing an aliphatic 'linker', that mimics the natural substrate galactose, and (iii) a long hydrophilic 'spacer'. The mimodye-ligands were immobilised to 1,1-carbonyldiimidazole-activated agarose chromatography support, via the spacer's terminal amino-group, to produce the respective mimodye adsorbents. Both immobilized mimodyes successfully bound P. fluorescens GaDH but failed to bind the enzyme from rabbit muscle. Adsorbent BM1 bound GaDH from green peas and Baker's yeast, but adsorbent BM2 failed to do so. The mimodye-ligand comprising D(+)-galactosamine (BM1), compared to BM2, exhibited higher purifying ability and enzyme recovery for P. fluorescens GaDH. The dissociation constants (KD) of BM1 and BM2 for P. fluorescens GaDH were determined by analytical affinity chromatography to be 5.9 microM and 15.4 microM, respectively. The binding capacities of adsorbents BM1 and BM2 were 18 U/mg adsorbent and 6 U/mg adsorbent, respectively. Adsorbents BM1 and BM2 were integrated in two different protocols for the purification P. fluorescens GaDH. Both protocols comprised as a common first step DEAE anion-exchange chromatography, with a second step of affinity chromatography on BM1 or BM2, respectively. The purified GaDH obtained from the protocols using BM1 and BM2 showed specific activities equal to 1077 and 854 U/mg, respectively. The former is the highest reported so far and the enzyme appeared as a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Galactosyl-mimodye ligands for Pseudomonas fluorescens beta-galactose dehydrogenase.

Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl-biomimetic dye ligands (galactosyl-mimodyes) for the target enzyme galactose dehydrogenase (GaDH). Using appropriate modelling methodology, a GaDH model was build based on a glucose-fructose oxidoreductase (GFO) protein template. Subsequent computational analysis predicted chimaeric mimodye-ligands comprising a NAD-pseudomimetic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose or shikimic acid) bearing an aliphatic 'linker' molecule. In addition, the designed mimodye ligands had an appropriate in length and chemical nature 'spacer' molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH. Following their synthesis, purification and analysis, the ligands were immobilized to agarose. The respective affinity adsorbents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescensbeta-galactose dehydrogenase. In addition, these mimodye affinity adsorbents displayed good selectivity, binding low amounts of enzymes other than GaDH. Further immobilized dye-ligands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromatographic behavior. Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purification of galactose dehydrogenase.

Galactosyl-biomimetic dye-ligands for the purification of Dactylium dendroides galactose oxidase.

Two anthraquinone galactosyl-biomimetic dye-ligands comprising, as terminal biomimetic moiety, galactose analogues (1-amino-1-deoxy-beta-D-galactose and D(+)-galactosamine) were designed for the enzyme galactose oxidase (GAO), using molecular modelling, synthesized and characterized. The biomimetic ligands were immobilized on agarose beads and the affinity adsorbents, together with a non-biomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their ability to purify GAO from Dactylium dendroides. Both biomimetic adsorbents showed higher purifying ability for GAO compared to the non-biomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising, as terminal biomimetic moiety, 1-amino-1-deoxy-beta-D-galactose (BM1) exhibited the highest purifying ability for GAO. This affinity adsorbent did not bind galactose dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, or glucose oxidase. The dissociation constant (K(D)) of the immobilized BM1 ligand with GAO was found to be equal to 45.8 microM, whereas the binding capacity was equal to 709 U per ml adsorbent. Therefore, the BMI adsorbent was integrated in a facile two-step purification procedure for GAO. The purified enzyme showed a specific activity equal to 2038 U/mg, the highest reported so far, approximately 74% overall recovery and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis.

Functional and structural roles of the glutathione-binding residues in maize (Zea mays) glutathione S-transferase I.

The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pK(a) of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser(11), His(40), Lys(41), Gln(53) and Ser(67)), which are located in the G-site, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steady-state kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues: (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH; (2) influence GSH thiol ionization, and thus its reactivity; (3) participate in k(cat) regulation by affecting the rate-limiting step of the reaction; and (4) in the cases of His(40), Lys(41) and Gln(53) play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 3(10)-helical segment of alpha-helix 2 (residues 35-46), as shown by limited proteolysis experiments. These structural perturbations are probably transmitted to the H-site through changes in Phe(35) conformation. This accounts for the modulation of K(CDNB)(m) by His(40), Lys(41) and Gln(53), and also for the intrasubunit communication between the G- and H-sites. Computer simulations using CONCOORD were applied to maize GST I monomer and dimer structures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found between the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 3(10)-helical segment of alpha-helix 2 (residues 35-46) undergoes the most significant structural rearrangements. These rearrangements are discussed in terms of enzyme catalytic mechanism.

The conserved Asn49 of maize glutathione S-transferase I modulates substrate binding, catalysis and intersubunit communication.

The functional and structural role of the conserved Asn49 of theta class maize glutathione S-transferase was investigated by site-directed mutagenesis. Asn49 is located in the type I beta turn formed by residues 49-52, and is involved in extensive hydrogen-bonding interactions between alpha helix 2 and the rest of the N-terminal domain. The substitution of Asn49 with Ala induces positive cooperativity for 1-chloro-2,4-dinitrobenzene (CDNB) binding as reflected by a Hill coefficient of 1.9 (S(0.5)CDNB = 0.43 mm). The positive cooperativity is also confirmed by following the isothermic binding of 1-hydroxyl-2,4-dinitrobenzene (HDNB) by UV-difference spectroscopy. In addition, the mutated enzyme exhibits: (a) an increase in the Km(GSH) value of about 6.5-fold, and decrease in kcat value of about fourfold; (b) viscosity-independent kinetic parameters; (c) lower thermostability, and (d) increased susceptibility to proteolytic attack by trypsin, when compared to the wild-type enzyme. It is concluded that Asn49 affects the rate-limiting step of the catalytic reaction, and contributes significantly to the structural and binding characteristics of both the glutathione binding site (G-site) and the electrophile substrate binding site (H-site) by affecting the structural integrity of a type I beta turn (comprising residues 49-52) and probably the flexibility of the highly mobile short 310 helical segment of alpha helix 2 (residues 35-46). These structural perturbations are probably transmitted, via Phe51 and Phe65, to alpha helix H3" of the adjacent subunit which contains key residues that interact with the electrophile substrate and contribute to the monomer-monomer contact region. This may accounts for the positive cooperativity observed.

New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes.

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140. Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GSTI. In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket. While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.

Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis.

The 2',3'-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mM for the fast phase, 0.45 mM for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min-1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme-oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme-oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the MODELLER 4 program. The model confirmed that Lys360 is positioned at the NAD+-binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360-->Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360-->Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.

Biomimetic dyes as affinity chromatography tools in enzyme purification.

Affinity adsorbents based on immobilized triazine dyes offer important advantages circumventing many of the problems associated with biological ligands. The main drawback of dyes is their moderate selectivity for proteins. Rational attempts to tackle this problem are realized through the biomimetic dye concept according to which new dyes, the biomimetic dyes, are designed to mimic natural ligands. Biomimetic dyes are expected to exhibit increased affinity and purifying ability for the targeted proteins. Biocomputing offers a powerful approach to biomimetic ligand design. The successful exploitation of contemporary computational techniques in molecular design requires the knowledge of the three-dimensional structure of the target protein, or at least, the amino acid sequence of the target protein and the three-dimensional structure of a highly homologous protein. From such information one can then design, on a graphics workstation, the model of the protein and also a number of suitable synthetic ligands which mimic natural biological ligands of the protein. There are several examples of enzyme purifications (trypsin, urokinase, kallikrein, alkaline phosphatase, malate dehydrogenase, formate dehydrogenase, oxaloacetate decarboxylase and lactate dehydrogenase) where synthetic biomimetic dyes have been used successfully as affinity chromatography tools.

Oxaloacetate decarboxylase from Pseudomonas stutzeri: purification and characterization.

Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245-fold to homogeneity from Pseudomonas stutzeri. The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity chromatography, and biomimetic-dye affinity chromatography. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monovalent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid. Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation.

Molecular modeling for the design of a biomimetic chimeric ligand. Application to the purification of bovine heart L-lactate dehydrogenase.

Molecular modeling was employed for the design of a biomimetic chimeric ligand for L-lactate dehydrogenase (LDH). This ligand is an anthraquinone monochlorotriazinyl dye comprising two moieties: (a) the ketocarboxyl biomimetic moiety, 2-(4-aminophenyl)-ethyloxamic acid, linked on the monochlorotriazine ring, mimicking the natural substrate of LDH, and (b) the anthraquinone chromophore moiety, linked also on the same monochlorotriazine ring via a diaminobenzenesulfonate group, acting as pseudomimetic of the cofactor NAD+. The positioning of the dye in the enzyme's binding site is primarily achieved by the recognition and positioning of the pseudomimetic anthraquinone moiety. The positioning of the biomimetic ketocarboxylic moiety is based on a match between the polar and hydrophobic regions of the enzyme's binding site with those of the biomimetic moiety of the ligand. The length of the biomimetic moiety is predetermined for the ketoacid to approach the enzyme catalytic site and form charge-charge interactions. The biomimetic chimeric ligand and the commercial nonbiomimetic ligand Cibacron(R) blue 3GA (CB3GA), were immobilized on crosslinked beaded agarose gel via their chlorotriazine ring. The two affinity adsorbents were evaluated for their purifying ability for LDH from six sources (bovine heart and pancreas, porcine muscle, chicken liver and muscle, and pea seeds). The biomimetic adsorbent exhibited approximately twofold higher purifying ability for LDH compared to the CB3GA adsorbent; therefore, the former was integrated in the purification procedure of LDH from bovine heart extract. The LDH afforded by this two-step purification procedure shows specific activity equal to 600 U/mg (25 degrees C) and a single band after SDS-PAGE analysis.

Chemical modification of barley root oxalate oxidase shows the presence of a lysine, a carboxylate, and disulfides, essential for enzyme activity.

Oxalate oxidase (OXO) was chemically modified using amino acid-specific reagents. The modification reactions were monitored spectrophotometrically, to follow the progress of labeling, and catalytically, to assess the effect of labeling on the enzyme function. The enzyme does not bear arginines essential for activity, since 2,3-butanedione and cyclohexanodione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Incubation of urea-pretreated OXO with N-acetylimidazole leads to labeling all 10 tyrosines without affecting the enzyme activity, thus suggesting that OXO does not have tyrosines essential for activity. However, OXO modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide followed by kinetic analysis, leads to the conclusion that the enzyme possesses one carboxylate essential for activity. When using the modifier 2,4, 6-trinitrobenzene sulfonic acid (TNBS), while 28 of the total 45 lysines are labeled within 3 h (the first 5 reacting lysines of the homopentametic enzyme are modified at a faster rate than the others), the enzyme rapidly loses 90% of its activity in the first 2 min, a period during which only one lysine is being labeled. Complete enzyme inactivation with TNBS is observed after approximately 8 min, when 5 lysines are being labeled. The modification of the first lysine also triggers the dissociation of native OXO to its subunits (after SDS-PAGE analysis), a phenomenon not observed with the other modifiers. These findings indicate that OXO bears a lysine per monomer, essential for enzyme activity. When using 5, 5-dithio-bis-(2-nitrobenzoic)acid to determine the number of disulfide bonds, in the presence of NaBH4, 10 sulfhydryls are determined, but in the absence of reducing agent, none are determined. Further, chloro-mercuribenzoate does not inactivate OXO but beta-mercaptoethanol does. Therefore, the sulfhydryls in OXO are not free but form disulfide bonds essential for activity. Furthermore, the metallo-chelating agents HgCl2 and 8-hydroxychinolin inactivate the enzyme, suggesting that barley root oxalate oxidase is a metalloenzyme. It is possible that the metal(s) are involved in the oxidative mechanism since the enzyme does not bear prosthetic groups such as FAD and FMN.

Purification of alcohol dehydrogenase from four genotypes of the olive fruit fly Bactrocera (Dacus) oleae.

This is the first report describing the purification of alcohol dehydrogenase (ADH) from four genotypes of the olive fruit fly Bactrocera oleae, the most important pest of olives in the Mediterranean region. The purified enzyme shows a single band after SDS-PAGE analysis, corresponding to subunit mass of 26 kDa. The native ADH shows a molecular mass of 48 kDa, after gel filtration HPLC analysis. The purification method incorporated a preliminary ammonium sulphate precipitation step, followed by an anion-exchange DEAE chromatography step, a dye affinity chromatography step on Cibacron blue 3GA, and an anion-exchange DEAE chromatography step employing the same column of the first step. The present method offers good overall recovery (40%) and high enzyme purity, and it is applicable to different genotypes. Furthermore, the method is rapid and economical, as it employs two cheap, widely used, and commercially available chromatography materials.

Oxalate oxidase from barley roots: purification to homogeneity and study of some molecular, catalytic, and binding properties.

Oxalate oxidase (OXO) was purified to homogeneity in three steps from roots of barley seedlings. The purification method comprised: (i) thermal treatment (60 degrees C, 10 min), (ii) affinity chromatography on immobilized either Procion turquoise MX-G dye or biomimetic aminoethyl oxamic blue dye, and (iii) affinity chromatography on immobilized lectin concanavalin A (overall performance: 1096-fold purification, 42% recovery). The purified enzyme has a specific activity of 34 U mg-1 (25 degrees C), and is a homopentamer of M(r) approximately 125,000 (HPLC analysis) showing a single band on SDS-polyacryl-amide gel electrophoresis (M(r) approximately 26,000) after staining with silver nitrate. The kinetic constants of the purified enzyme for oxalate are K(m) 0.27 mM and kcat 22 s-1 (37 degrees C), whereas at [oxalate] > or = 4 mM the enzyme exhibited substrate inhibition. Barley root OXO contains no prosthetic group absorbing at 370 or 450 nm, and riboflavin and FAD have no effect on its activity. The enzyme is activated by 1 mM each of Ca2+ (1.7-fold) and Pb2+ (2.6-fold). Irreversible inactivation studies with denatured (70 degrees C) and native (37 degrees C) enzyme using the sulfhydryl-attacking reagent 5,5-dithiobis(2-nitrobenzoic) acid (1.4 mM), in the presence and absence of SDS, respectively, have shown that denatured OXO (4% SDS, 10 min, 100 degrees C) exhibited 10 HS groups per molecule, whereas native OXO displayed one accessible HS group per molecule after approximately 15 min incubation and, over the same period, maintained its catalytic activity to 90%. Furthermore, native OXO treated with beta-mercaptoethanol (1 mM) lost 83% of its catalytic activity within 5 min. These findings indicate that some cysteines may preserve the catalytic activity of OXO by maintaining the integrity of its tertiary structure via disulfide bond formation.

L-Malate dehydrogenase from Pseudomonas stutzeri: purification and characterization.

L-Malate dehydrogenase (MDH) from Pseudomonas stutzeri was purified to homogeneity by a two-step procedure comprising anion-exchange chromatography and affinity chromatography on immobilized anthraquinone alpha-ketocarboxyl biomimetic dye. The enzyme has molecular mass of 66,500 Da and consists of two identical subunits of molecular mass of approximately 34,000 Da. Initial velocity, product inhibition, and binding studies were consistent with an ordered Bi-Bi mechanism for the enzyme action and the formation of a ternary complex. The enzyme is susceptible to activation and inhibition by its substrates. Thermodynamic analysis and kinetic inhibition studies were performed for determining basic equilibrium and kinetic constants. Malate dehydrogenase was covalently inactivated by a dichlorotriazine dye, Vilmafix Blue A-R (VBAR). The inactivation process follows first-order kinetics, and the results from kinetic analysis suggested the formation of a noncovalent enzyme-dye complex prior to the covalent reaction, with Kd 84.6 microM and a maximum rate constant 0.16 min(-1). The enzyme inactivation process was partially inhibited by substrates and inhibitors. Quantitatively inactivated MDH contained approximately 1 mole of dye per mole of enzyme subunit. The denatured enzyme contains 10 sulfhydryl groups per subunit, as shown after reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid), of which 5 can be titrated also in the native enzyme, exhibiting time-dependent reactivity. One sulfhydryl group is located in the coenzyme binding site. This study shows that the physical and catalytic properties of P. stutzeri MDH strongly resemble those of the mitochondrial eukariotic enzyme. This finding strengthens the existing view that, in the evolution process, the mitochondrial MDH might have appeared before the cytoplasmic.

Design and study of peptide-ligand affinity chromatography adsorbents: application to the case of trypsin purification from bovine pancreas.

The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C-terminal (site P(1)) for trypsin bio-recognition, a Pro or Ala in site P(2), and a Thr or Val in site P(3). Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 micromol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 micromol/g gel. The K(D) values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The K(D) values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (K(D) 8.7 microM), whereas the sequence H-TAR-OH displayed the lowest (K(D) 38 microM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 micromol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. (c) 1997 John Wiley & Sons, Inc.

Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site.

The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 microM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217.

Molecular modelling for the design of chimaeric biomimetic dye-ligands and their interaction with bovine heart mitochondrial malate dehydrogenase.

Molecular modelling and kinetic inhibition studies, as well as KD determinations by both difference-spectra and enzyme-inactivation studies, were employed to assess the ability of purpose-designed chimaeric biomimetic dyes (BM dyes) to act as affinity ligands for bovine heart L-malate dehydrogenase (MDH). Each BM dye was composed of two enzyme-recognition moieties. The terminal biomimetic moiety bore a carboxyl or a keto acid structure linked to the triazine ring, thus mimicking the substrate of MDH. The chromophore anthraquinone moiety remained unchanged and the same as that of the parent dye Vilmafix Blue A-R (VBAR), recognizing the nucleotide-binding site of MDH. The monochlorotriazine BM dyes did not inactivate MDH but competitively inhibited inactivation by the parent dichlorotriazine dye VBAR. Dye binding to MDH was accompanied by a characteristic spectral change in the range 500-850 nm. This phenomenon was reversed after titration with increasing amounts of NADH. When compared with VBAR, Cibacron Blue 3GA and two control non-biomimetic anthraquinone dyes, all BM dyes exhibited lower KD values and therefore higher affinity for MDH. The enzyme bound preferably to BM ligands substituted with a biomimetic aromatic moiety bearing an alpha-keto acid group and an amide linkage, rather than a monocarboxyl group. Thus the biomimetic dye bearing p-aminobenzyloxanilic acid as its terminal biomimetic moiety (BM5) exhibited the highest affinity (KD 1.3 microM, which corresponded to a 219-fold decrease over the KD of a control dye). BM5 displayed competitive inhibition with respect to both NADH (Ki 2.7 microM) and oxaloacetate (Ki 9.6 microM). A combination of molecular modelling and experimental studies has led to certain conclusions. The positioning of the dye in the enzyme is primarily achieved by the recognition and positioning of the nucleotide-pseudomimetic anthraquinone moiety. The hydrophobic groups of the dye provide the driving force for positioning of the ketocarboxyl biomimetic moiety. A match between the alternating polar and hydrophobic regions of the enzyme binding site with those of the biomimetic moiety is desirable. The length of the biomimetic moiety should be conserved in order for the keto acid to approach the enzyme active site and form charge-charge interactions.