Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg --> Ala), K107A (Lys --> Ala), and R148A (Arg --> Ala), had minimal actin binding activity relative to wild type (wt) aldolase, and one mutant, K229A (Lys --> Ala), had intermediate actin binding activity. A mutant with approximately 4,000-fold reduced catalytic activity, D33S (Asp --> Ser), had normal actin binding activity. The aldolase substrates and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and dihydroxyacetone phosphate, reversed the gelling of wt aldolase and F-actin, consistent with at least partial overlap of catalytic and actin-binding sites on aldolase. Molecular modeling reveals that the actin-binding residues we have identified are clustered in or around the catalytic pocket of the molecule. These data confirm that the aldolase-actin interaction is due to specific binding, and they suggest that electrostatic interactions between specific residues, rather than net charge, mediate this interaction. Low concentration of wt and D33S aldolase caused formation of high viscosity actin gel networks, while high concentrations of wt and D33S aldolase resulted in solation of the gel by bundling actin filaments, consistent with a potential role for this enzyme in the regulation of cytoplasmic structure.
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PMID:The molecular nature of the F-actin binding activity of aldolase revealed with site-directed mutants. 863 11

Cathepsin B was isolated from buffalo liver by salt fractionation, ion-exchange resin treatment, gel filtration and repeated ion-exchange chromatography using a linear salt gradient. The enzyme showed activity, against denatured hemoglobin (or ovalbumin), alpha-N-benzoyl-DL-arginine p-nitroanilide (BAPNA), and alpha-benzoyl-DL-arginine-naphthylamine (BANA). It inactivated buffalo muscle aldolase with a half life period of 21 min. The pH-activity profiles obtained for the digestion of hemoglobin (or ovalbumin) and aldolase inactivation by the enzyme were found to be different. The enzyme (mol wt 27,800 by SDS-PAGE) eluted in gel filtration with a molecular weight of 27,000 and a Stokes radius of 2.31 nm. The results showed buffalo cathepsin B to be a single-chain molecule. The N- and C-terminal amino acids of the enzyme were found to be leucine and aspartic acid, respectively. It contained 0.7% concanavalin A reactive neutral carbohydrate. The amino acid composition of buffalo cathepsin B was found to be similar to that of human liver cathepsin B. The optical properties of the buffalo enzyme were found consistent with its aromatic amino acid content. The isoionic pH of the enzyme was found to be 5.70 and the intrinsic viscosity was 3.48 ml/g whence the frictional ratio, f/f0 was computed to be 1.10 suggesting that the native enzyme conformation is compact and is globular in solution.
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PMID:Isolation, purification and properties of cathepsin B from buffalo liver. 893 19

Although airborne allergens in bakeries and confectioneries cause one of the most common forms of occupational asthma, namely, bakers' asthma, only a few of them are known in detail so far. Here we summarize current knowledge of bakery allergens and describe our own two-dimensional (2-D) immunoelectrophoresis studies of wheat-flour allergens as well as the allergenic baking enzyme Asp o 2. Out of approximately 700 soluble wheat polypeptides, 70 show IgE binding; the following wheat-flour allergens could be identified and characterized: members of the alpha-amylase inhibitor family (14-18 kDa), acyl-CoA oxidase (26 kDa), peroxidase (36 kDa), and fructose-bisphosphate aldolase (37 kDa). However, the great majority of the soluble wheat-flour allergens, mainly located in the 27-, 55-, and 70-kDa areas of the 2-D immunoblots with pI values of 5.8-6.8, 5.9-6.5, and 5.5-6.1, respectively, are not known at present. Asp o 2, to which approximately 25% of all bakers with respiratory symptoms are sensitized, is a well-characterized starch-cleaving enzyme. We conclude that great effort is still needed to describe all major wheat-flour allergens. As shown by Asp o 2, knowledge of the causative allergens and their characteristics enables us to initiate very effective preventive measures such as the introduction of granulated allergenic products.
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PMID:Characterized allergens causing bakers' asthma. 968 37

Compartmentation of proteins in cells is important to proper cell function. Interactions of F-actin and glycolytic enzymes is one mechanism by which glycolytic enzymes can compartment. Brownian dynamics (BD) simulations of the binding of the muscle form of the glycolytic enzyme fructose-1,6-bisphosphate aldolase (aldolase) to F- or G-actin provide first-encounter snapshots of these interactions. Using x-ray structures of aldolase, G-actin, and three-dimensional models of F-actin, the electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations provided solution complexes of aldolase with F- or G-actin. All complexes demonstrate the close contacts between oppositely charged regions of the protein surfaces. Positively charged surface regions of aldolase (residues Lys 13, 27, 288, 293, and 341 and Arg 257) are attracted to the negatively charged amino terminus (Asp 1 and Glu 2 and 4) and other patches (Asp 24, 25, and 363 and Glu 361, 364, 99, and 100) of actin subunits. According to BD results, the most important factor for aldolase binding to actin is the quaternary structure of aldolase and actin. Two pairs of adjacent aldolase subunits greatly add to the positive electrostatic potential of each other creating a region of attraction for the negatively charged subdomain 1 of the actin subunit that is exposed to solvent in the quaternary F-actin structure.
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PMID:Brownian dynamics simulations of interactions between aldolase and G- or F-actin. 987 19

Proposing that a blend of the chemical diversity of small synthetic molecules with the immunological characteristics of the antibody molecule will lead to therapeutic agents with superior properties, we here present a device that equips small synthetic molecules with both effector function and long serum half-life of a generic antibody molecule. As a prototype, we developed a targeting device that is based on the formation of a covalent bond of defined stoichiometry between a 1,3-diketone derivative of an integrin alpha(v)beta(3) and alpha(v)beta(5) targeting Arg-Gly-Asp peptidomimetic and the reactive lysine of aldolase antibody 38C2. The resulting complex was shown to (i) spontaneously assemble in vitro and in vivo, (ii) selectively retarget antibody 38C2 to the surface of cells expressing integrins alpha(v)beta(3) and alpha(v)beta(5), (iii) dramatically increase the circulatory half-life of the Arg-Gly-Asp peptidomimetic, and (iv) effectively reduce tumor growth in animal models of human Kaposi's sarcoma and colon cancer. This immunotherapeutic has the potential to target a variety of human cancers, acting on both the vasculature that supports tumor growth as well as the tumor cells themselves. Further, by use of a generic antibody molecule that forms a covalent bond with a 1,3-diketone functionality, essentially any compound can be turned into an immunotherapeutic agent thereby not only increasing the diversity space that can be accessed but also multiplying the therapeutic effect.
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PMID:Chemically programmed monoclonal antibodies for cancer therapy: adaptor immunotherapy based on a covalent antibody catalyst. 1270 56

Malaria remains a major disease of mankind, and resistance to existing therapeutics is rapidly emerging. Limited financial investment to develop new therapeutics requires the careful selection of well-defined targets from the causative parasite, Plasmodium falciparum. In these circumstances, protein crystallography can provide valuable structural detail to facilitate both the selection of suitable targets and the development of compounds to provide novel drug candidates. This review summarises the current involvement of crystallographic studies in anti-malarial drug development programmes. Protein crystallography is increasingly central to the exploitation of a number of potential Plasmodial targets. including the aspartic acid proteases (plasmepsins) and cysteine proteases (falcipains) involved in haem degradation within the parasite food vacuole. Lead compounds are being identified from collections previously synthesised against homologous human enzymes. Plasmodium have an unusual dependence on the glycolytic pathway relative to their human hosts, and this is reflected in subtle structural differences identified in the crystal structures of a number of parasite glycolytic enzymes including aldolase and lactate dehydrogenase. Other enzymes from a range of biosynthetic pathways have also been targeted in crystallographic studies. These include dihydrofolate reductase, the target of existing anti-folate therapeutics, and enoyl reductase from the fatty acid biosynthesis pathway which is already the target of effective bacteriocides. Crystal structures of these drug-enzyme complexes not only allow visualisation and improvement of inhibitor-protein contacts, but in the former case have also been used to probe the molecular basis of emerging anti-malarial drug resistance. Crystallography is similarly proving valuable as a tool to facilitate the development of inhibitors of purine salvage, isoprenoid synthesis and utilisation, and protein processing mechanisms.
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PMID:Structure-based approaches to the development of novel anti-malarials. 1501 47

The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine. Using active site mutants of FBPA I from Thermoproteus tenax, we have solved the crystal structures of the enzyme covalently bound to the carbinolamine of the substrate fructose 1,6-bisphosphate and noncovalently bound to the cyclic form of the substrate. The structures, determined at a resolution of 1.9 A and refined to crystallographic R factors of 0.148 and 0.149, respectively, represent the first view of any FBPA I in these two stages of the reaction pathway and allow detailed analysis of the roles of active site residues in catalysis. The active site geometry of the Tyr146Phe FBPA variant with the carbinolamine intermediate supports the notion that in the archaeal FBPA I Tyr146 is the proton donor catalyzing the conversion between the carbinolamine and Schiff base. Our structural analysis furthermore indicates that Glu187 is the proton donor in the eukaryotic FBPA I, whereas an aspartic acid, conserved in all FBPA I enzymes, is in a perfect position to be the general base facilitating carbon-carbon cleavage. The crystal structure of the Trp144Glu, Tyr146Phe double-mutant substrate complex represents the first example where the cyclic form of beta-fructose 1,6-bisphosphate is noncovalently bound to FBPA I. The structure thus allows for the first time the catalytic mechanism of ring opening to be unraveled.
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PMID:Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates. 1576 50

Interactions of phosphate derivatives of 2,6-dihydroxynaphthalene (NA-P(2)) and 1,6-dihydroxy-2-naphthaldehyde (HNA-P, phosphate at position 6) with fructose-1,6-bisphosphate aldolase from rabbit muscle were analyzed by enzyme kinetics, difference spectroscopy, site-directed mutagenesis, mass spectrometry, and molecular dynamics. Enzyme activity was competitively inhibited by NA-P(2), whereas HNA-P exhibited slow-binding inhibition with an overall inhibition constant of approximately 24 nM. HNA-P inactivation was very slowly reversed with t(1/2) approximately 10 days. Mass spectrometry and spectrophotometric absorption indicated that HNA-P inactivation occurs by Schiff base formation. Rates of enzyme inactivation and Schiff base formation by HNA-P were identical and corresponded to approximately 4 HNA-P molecules bound par aldolase tetramer at maximal inhibition. Site-directed mutagenesis of conserved active site lysine residues 107, 146, and 229 and Asp-33 indicated that Schiff base formation by HNA-P involved Lys-107 and was promoted by Lys-146. Titration of Lys-107 by pyridoxal 5-phosphate yielded a microscopic pK(a) approximately 8 for Lys-107, corroborating a role as nucleophile at pH 7.6. Site-directed mutagenesis of Ser-271, an active site residue that binds the C(1)-phosphate of dihydroxyacetone phosphate, diminished HNA-P binding and enabled modeling of HNA-P in the active site. Molecular dynamics showed persistent HNA-P phosphate interactions with the C(1)-phosphate binding site in the noncovalent adduct. The naphthaldehyde hydroxyl, ortho to the HNA-P aldehyde, was essential for promoting carbinolamine precursor formation by intramolecular catalysis. The simulations indicate a slow rate of enzyme inactivation due to competitive inhibition by the phenate form of HNA-P, infrequent nucleophilic attack in the phenol form, and significant conformational barrier to bond formation as well as electrostatic destabilization of protonated ketimine intermediates. Solvent accessibility by Lys-107 Nz was reduced in the covalent Schiff base complex, and in those instances where water molecules interacted with Lys-107 in the simulations, Schiff base hydrolysis was not mechanistically favorable. The findings at the molecular level corroborate the observed mechanism of slow-binding tight inhibition by HNA-P of muscle aldolase and should serve as a blueprint for future aldolase inhibitor design.
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PMID:Hydroxynaphthaldehyde phosphate derivatives as potent covalent Schiff base inhibitors of fructose-1,6-bisphosphate aldolase. 1580 36

Monoclonal antibody (mAb) 38C2 belongs to a group of catalytic antibodies that were generated by reactive immunization and contains a reactive lysine. 38C2 catalyzes aldol and retro-aldol reactions, using an enamine mechanism, and mechanistically mimics natural aldolase enzymes. In addition, mAb 38C2 can be redirected to target integrins alpha(v)beta(3) and alpha(v)beta(5) through the formation of a covalent bond between a beta-diketone derivative of an arginine-glycine-aspartic acid (RGD) peptidomimetic and the reactive lysine residue in the antibody combining site to provide the chemically programmed mAb cp38C2. In this study, we investigated the potential of enhancing the activity of receptor-binding small molecule drug (SCS-873) through antibody conjugation. Using a M21 human melanoma xenograft model in nude mice, cp38C2 inhibited the growth of the tumor by 81%. The chemically programmed antibody was shown to be highly active at a low concentration while SCS-873 alone was ineffective even at dosages 1,000-fold higher than those used for the chemically programmed antibody. In vitro programming of the catalytic antibody was shown to be as effective as in vivo programming. In an experimental metastasis assay, treatment with mAb cp38C2 significantly prolonged overall survival of tumor-bearing severe combined immuno-deficient (SCID) mice when compared to treatment with unprogrammed mAb 38C2, SCS-873 alone or the integrin-specific monoclonal antibody LM609. In vitro, cp38C2 inhibited human and mouse endothelial and human melanoma cell adhesion, migration and invasion. Additionally, cp38C2 inhibited human and mouse endothelial cell proliferation and was active in complement-dependent cytotoxicity assays. These studies establish the potential of chemically programmed monoclonal antibodies as a novel and effective class of immunotherapeutics that combine the merits of traditional small molecule drug design with immunotherapy.
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PMID:Small molecule drug activity in melanoma models may be dramatically enhanced with an antibody effector. 1657 Feb 83

A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 A) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds.
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PMID:Modeling the interaction between aldolase and the thrombospondin-related anonymous protein, a key connection of the malaria parasite invasion machinery. 1715 57


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