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)

Antibodies that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction, have been generated. The mechanism for antibody catalysis of this reaction mimics that used by natural class I aldolase enzymes. Immunization with a reactive compound covalently trapped a Lys residue in the binding pocket of the antibody by formation of a stable vinylogous amide. The reaction mechanism for the formation of the covalent antibody-hapten complex was recruited to catalyze the aldol reaction. The antibodies use the epsilon-amino group of Lys to form an enamine with ketone substrates and use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The antibodies control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.
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PMID:Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. 852 66

Human erythrocyte band 3 inhibits glycolytic enzymes, including aldolase, by binding these cytoplasmic enzymes at its N-terminus. Phosphorylation of Y8 disrupts inhibition, and there is evidence that in vivo glycolysis levels in erythrocytes are regulated in part by a phosphorylation/dephosphorylation signaling pathway. The structural basis for control by phosphorylation has been investigated by NMR studies on a complex between aldolase and a synthetic peptide corresponding to the first 15 residues of band 3 (MEELQDDYEDMMEEN-NH2). The structure of this band 3 peptide (B3P) when it is bound to rabbit muscle aldolase was determined using the exchange-transferred nuclear Overhauser effect (ETNOE). Two hundred NMR structures for B3P were generated by simulated annealing molecular dynamics with NMR-derived distance restraints and excluding electrostatic terms. Twenty structures were further refined against a force field including full partial charges. The important conformational feature of B3P in the bound state is a folded loop structure involving residues 4-9 and M12 that surrounds Y8 and is stabilized by a hydrophobic cluster with the ring of Y8 sandwiched between the methyl groups of L4 and M12. Differential line broadening indicates that this loop structure binds aldolase in a relatively specific manner, while terminal regions are structurally heterogeneous. To better understand B3P inhibition of aldolase and the mechanism of phosphorylation control, a complex was modeled by docking B3P into the active site of aldolase and optimizing the fit using restrained molecular dynamics and energy minimization. The B3P loop is complementary in conformation to the beta-barrel central core containing the aldolase active site residues. Binding is electrostatic in nature with numerous ionic and hydrogen-bonding interactions involving several conserved lysine and arginine residues of aldolase. How phosphorylation of band 3 could disrupt inhibition was considered by modeling a phosphoryl moiety onto Y8 of B3P. An energetic analysis with respect to rigid phosphate rotation suggests that aldolase inhibition is reversed primarily because of electrostatic repulsion between B3P residues that destabilizes the B3P loop formed in the complex. This proposed intramolecular mechanism for blocking protein--protein association by electrostatic repulsion with the phosphoryl group may be applicable to other protein--protein signaling complexes.
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PMID:Solution structure of a band 3 peptide inhibitor bound to aldolase: a proposed mechanism for regulating binding by tyrosine phosphorylation. 852 30

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

The process of reactive immunization has been used to induce efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes. Reactive immunogens are those that react chemically during induction of the immune response. This same reaction is used later in catalysis. In essence one immunizes with the equivalent of a mechanism-based inhibitor. The difference is that instead of inhibiting a mechanism, a mechanism is induced. This advance allows the experimenter to dictate the exact mechanism by which catalytic antibodies proceed. The hapten used in the present study is a 1,3-diketone that both traps the requisite lysine residue to initiate formation of the enamine and induces a binding pocket that overcomes the entropic barrier of this bimolecular reaction.
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PMID:Using the process of reactive immunization to induce catalytic antibodies with complex mechanisms: aldolases. 875 55

Sialate lyase (sialate aldolase; systematic name N-acetylneuraminate pyruvate-lyase, EC 4.1.3.3) was isolated as soluble enzyme from pig kidney and purified 630-fold using a heating step, gel filtration, and chromatography on immobilized neuraminic acid beta-methyl glycoside in 14% yield to apparent homogeneity as tested by SDS-gel electrophoresis. The molecular mass is 58 kDa and the pH-optimum is at pH 7.2. Kinetic parameters were determined with N-acetyl-neuraminic acid as substrate: Km 3.7 mM and Vmax 37.1 mU. The lyase cleaves only free sialic acids with relative rates of 100% for N-acetylneuraminic acid, 55% for N-glycolylneuraminic acid and 32% for N-acetyl-9-O-acetylneuraminic acid, whereas N-acetyl-4-O-acetylneuraminic acid or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid are not substrates. Enzyme activity was inhibited with p-chloromercuribenzoate, o-phenanthroline, cyanide, 5-diazonium-1-H-tetrazole, 5,5'-dithiobis(2-nitrobenzoic acid), diethylpyro-carbonate, and Rose Bengal in the presence of light and O2. Reduction with sodium borohydride in the presence of N-acetylneuraminic acid or pyruvate resulted in irreversible inhibition of enzyme activity. The inhibition experiments suggest the involvement of histidine, lysine and SH-residues in enzyme catalysis. Thus, this mammalian lyase most probably belongs to the Class I aldolases, and has properties similar to the same enzyme from Clostridium perfringens and is active with the alpha-form of N-acetylneuraminic acid.
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PMID:Isolation and characterization of sialate lyase from pig kidney. 882 20

We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme. To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.
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PMID:L-allo-threonine aldolase from Aeromonas jandaei DK-39: gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis. 917

The interactions of the phosphorylated derivatives of hydroquinone (HQN-P2), resorcinol (RSN-P2), 4-hydroxybenzaldehyde (HBA-P) and 2, 4-dihydroxybenzaldehyde (DHBA-P; phosphate group at position 4) with fructose bisphosphate aldolase were analysed by enzyme kinetics, UV/visible difference spectroscopy and site-directed mutagenesis. Enzyme activity was competitively inhibited in the presence of HQN-P2, RSN-P2 and HBA-P, whereas DHBA-P exhibited slow-binding inhibition. Inhibition by DHBA-P involved active-site Schiff-base formation and required a phenol group ortho to the aldehyde moiety. Rates of enzyme inactivation and of Schiff-base formation by DHBA-P were identical, and corresponded to 3.2-3.5 DHBA-P molecules covalently bound per aldolase tetramer at maximal inactivation. Site-directed mutagenesis of the active-site lysine residues at positions 107, 146 and 229 was found to be consistent with Schiff-base formation between DHBA-P and Lys-146, and this was promoted by Lys-229. Mutation of Glu-187, located vicinally between Lys-146 and Lys-229 in the active site, perturbed the rate of Schiff-base formation, suggesting a functional role for Glu-187 in Schiff-base formation and stabilization. The decreased cleavage activity of the active-site mutants towards fructose 1, 6-bisphosphate is consistent with a proton-transfer mechanism involving Lys-229, Glu-187 and Lys-146.
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PMID:Inhibition of rabbit muscle aldolase by phosphorylated aromatic compounds. 917 4

Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.
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PMID:Immune versus natural selection: antibody aldolases with enzymic rates but broader scope. 940 38

Unknown proteins isolated from mutant tissues of rice (Oryza sativa L.) recovered from inhibitor selections were subsequently peptide microsequenced. Database searches putatively identified one peptide as fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Tissues of mutant rice, PI564784, and wild type (cv Calrose 76) tissues were evaluated for aldolase activity. Total enzyme activities were slightly lower in the mutant than the control but the differences were not significant. Although the mutant phenotype is for enhanced lysine and protein, we ascribe the small aldolase differences to physiological adjustments, rather than to DNA modifications of the aldolase gene(s). Homologies of rice peptides with aldolases from a range of species, as well as rice cell culture expressed sequence tags (ESTs) are presented. Some amino acids sequences are highly conserved. The mutant phenotype expressing stress proteins is not likely to be defined by a change in rice aldolases.
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PMID:Fructose 1,6-bisphosphate aldolase activity in leaves of a rice mutant selected for enhanced lysine. 941 99

A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L specific at the alpha position, whereas it cannot distinguish between threo and erythro forms at the beta position. In addition to threonine, the enzyme also acts on various other L-beta-hydroxy-alpha-amino acids, including L-beta-3,4-dihydroxyphenylserine, L-beta-3,4-methylenedioxyphenylserine, and L-beta-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity L-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the L-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5'-phosphate of the enzyme to catalyze the reversible aldol reaction.
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PMID:Gene cloning, nucleotide sequencing, and purification and characterization of the low-specificity L-threonine aldolase from Pseudomonas sp. strain NCIMB 10558. 946 92


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