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)

Two sensitive assays, one which fluorometrically measures only the L isomer of 2-keto-4-hydroxyglutarate after decarboxylation to L-malate and the other which spectrophotometrically determines both enantiomers by reductive amination with glutamate dehydrogenase, are described. By use of these assays, the steady-state kinetics of the aldol condensation of pyruvate with glyoxylate, as catalyzed by 2-keto-4-hydroxyglutarate aldolase from either bovine liver or Escherichia coli, were studied as was the inhibition of this reaction by glyoxylate and other anions. For the E. coli aldolase, double-reciprocal plots are linear except at high (above 5 mM) glyoxylate concentrations; apparent Km values increase with increasing concentrations of the fixed substrate. The data are consistent with an ordered reaction sequence. Inhibition by halides follows the lyotropic or Hofmeister series. Esters are not good inhibitors; mono-, di-, and tricarboxylic acids are increasingly inhibitory. Of the substrate analogues tested, hydroxypyruvate is the most potent inhibitor. Inhibition studies with citrate, acetaldehyde, and glyoxylate (all competitive inhibitors) suggest there are two domains at the active site-the Schiff base forming lysyl residue which interacts with carbonyl analogues (like acetaldehyde) and a center of positive charge which binds anions (like citrate). In contrast to the bacterial enzyme, liver 2-keto-4-hydroxyglutarate aldolase is inhibited in a competitive manner by much lower concentrations (0.1 mM or even lower) of glyoxylate. Many salts and some carboxylic acids activate the liver enzyme. Similarly, substrate analogues like 2-ketobutyrate and fluoropyruvate are mild activators; no effect is seen with acetaldehyde. Besides glyoxylate, only glyoxal, 2-ketoglutarate, and hydroxypyruvate inhibit the aldol condensation reaction. A uniform value of 1 is found for the number of inhibitor molecules bound per active site of either liver or E. coli 2-keto-4-hydroxyglutarate aldolase.
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PMID:Steady-state kinetics and inhibition studies of the aldol condensation reaction catalyzed by bovine liver and Escherichia coli 2-keto-4-hydroxyglutarate aldolase. 701 77

Spontaneous mutants which acquired the ability to utilize d-allylglycine (d-2-amino-4-pentenoic acid) and dl-cis-crotylglycine (dl-2-amino-cis-4-hexenoic acid) but not l-allylglycine or dl-trans-crotylglycine could be readily isolated from Pseudomonas putida mt-2 (PaM1). Derivative strains of PaM1 putatively cured of the TOL (pWWO) plasmid were incapable of forming mutants able to utilize the amino acids for growth; however, this ability could be regained by conjugative transfer of the TOL (pWWO) plasmid from a wild-type strain of mt-2 or of the TOL (pDK1) plasmid from a related strain of P. putida (HS1), into cured recipients. dl-Allylglycine-grown cells of one spontaneous mutant (PaM1000) extensively oxidized dl-allylglycine and dl-cis-crotylglycine, whereas only a limited oxidation was observed toward l-allylglycine and dl-trans-crotylglycine. Cell extracts prepared from PaM1000 cells contained high levels of 2-keto-4-hydroxyvalerate aldolase and 2-keto-4-pentenoic acid hydratase, the latter enzyme showing higher activity toward 2-keto-cis-4-hexenoic acid than toward the trans isomer. Levels of other enzymes of the TOL degradative pathway, including toluate oxidase, catechol-2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-hydroxymuconic semialdehyde dehydrogenase, were also found to be elevated after growth on allylglycine. Whole cells of a putative cured strain, PaM3, accumulated 2-keto-4-pentenoic acid from d-allylglycine, which was shown to be rapidly degraded by cell extracts of PaM1000 grown on dl-allylglycine. These same cell extracts were also capable of catalyzing the dehydrogenation of d- but not l-allylglycine and were further found to metabolize the amino acid completely to pyruvate and acetaldehyde. Differential centrifugation of crude cell extracts localized d-allylglycine dehydrogenase activity to membrane fractions. The results are consistent with a catabolic pathway for d-allylglycine and dl-cis-crotylglycine involving the corresponding keto-enoic acids as intermediates, the further metabolism of which is effected by the action of TOL plasmid-encoded enzymes.
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PMID:Metabolism of allylglycine and cis-crotylglycine by Pseudomonas putida (arvilla) mt-2 harboring a TOL plasmid. 728 32

The todFC1C2BADE gene cluster in Pseudomonas putida F1 encodes enzymes for the first four steps of toluene degradation, leading to the formation of 2-hydroxypenta-2,4-dienoate (HPD). Here, we report the nucleotide (nt) sequence and expression of the remaining three genes of the tod pathway, downstream from todE and arranged in the order, todGIH. The deduced amino acid (aa) sequences of TodG [HPD hydratase (268 aa)], TodH [4-hydroxy-2-oxovalerate (HO) aldolase (352 aa)] and TodI [acylating aldehyde (AA) dehydrogenase (316 aa)] are compared with the isofunctional proteins present in the meta-cleavage pathways of other bacteria. New sequence motifs are identified. The highly conserved TodH and TodI sequences are potentially useful DNA probes for biomonitoring purposes.
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PMID:Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1. 806 6

From a soil isolate, Pseudomonas strain C18, we cloned and sequenced a 9.8-kb DNA fragment that encodes dibenzothiophene-degrading enzymes. Nine open reading frames were identified and designated doxABDEFGHIJ. Collectively, we refer to these genes as the DOX pathway. At the nucleotide level, doxABD are identical to the ndoABC genes that encode naphthalene dioxygenase of Pseudomonas putida. The DoxG protein is 97% identical to NahC (1,2-dihydroxynaphthalene dioxygenase) of P. putida. DoxE has 37% identity with cis-toluene dihydrodiol dehydrogenase. DoxF is similar to the aldehyde dehydrogenases of many organisms. The predicted DoxHIJ proteins have no obvious sequence similarities to known proteins. Gas chromatography with a flame ionization detector and mass spectroscopy confirmed that the DOX proteins convert naphthalene to salicylate and converting phenanthrene to 1-hydroxy-2-naphthoic acid. doxI mutants convert naphthalene to trans-o-hydroxybenzylidenepyruvate, indicating that the DoxI protein is similar to NahE (trans-o-hydroxybenzylidenepyruvate hydratase-aldolase). Comparison of the DOX sequence with restriction maps of cloned naphthalene catabolic pathway (NAH) genes revealed many conserved restriction sites. The DOX gene arrangement is identical to that proposed for NAH, except that the NAH equivalent of doxH has not been recognized. DoxH may be involved in the conversion of 2-hydroxy-4-(2'-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoat e to cis-o-hydroxybenzylidenepyruvate. doxJ encodes an enzyme similar to NahD (isomerase). Our findings indicate that a single genetic pathway controls the metabolism of dibenzothiophene, naphthalene, and phenanthrene in strain C18 and that the DOX sequence encodes a complete upper naphthalene catabolic pathway similar to NAH.
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PMID:Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. 822 31

2-Amino-3-ketobutyrate ligase catalyzes the reversible, pyridoxal 5'-phosphate-dependent condensation of glycine with acetyl CoA forming the unstable intermediate, 2-amino-3-ketobutyrate. Several independent lines of evidence indicate that the pure protein obtained in the purification of this ligase from Escherichia coli also has L-threonine aldolase activity. The evidence includes: (a), a constant ratio of specific activities (aldolase/ligase) at all stages of purifying 2-amino-3-ketobutyrate ligase to homogeneity; (b), the same rate of loss of aldolase and ligase activities during controlled heat inactivation of the pure protein at 60 degrees C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymatic activities that are not significantly different during slow inactivation by iodoacetamide, with and without L-threonine added; (d), coincident rates of loss and essentially identical rates of recovery of aldolase activity and ligase activity during resolution of the holoenzyme with hydroxylamine followed by reconstitution with pyridoxal 5'-phosphate. No aldolase activity is observed with D-threonine as substrate and L-allothreonine is about 25% as effective as L-threonine. Whereas ligase activity has a sharp pH optimum at 7.5, the aldolase activity of this pure protein is maximal at pH 9.0. Comparative apparent Km values for glycine (ligase) and L-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 mumol of CoA released/min per mg vs. 0.014 mumol of acetaldehyde formed (NADH oxidized)/min per mg.
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PMID:Identity and some properties of the L-threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli. 834 29

The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp. strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) [acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10]. A procedure for purifying these two enzyme activities to homogeneity is reported here. The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa. Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes. The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex. In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol. 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures. The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.
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PMID:Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600. 841 88

Sequence homology among nonconserved residues 357-362 of the COOH-terminal region in fructose-1,6-bisphosphate aldolases correlates with isozyme classification of aldolases. Recombinant chimers of human liver and maize aldolases were constructed by exchanging residues 357-362 with those from muscle, maize, and liver isozyme and by insertion in the maize sequence at position 349 rabbit muscle and liver residues 346-349. Activity variation among the chimers relative to native controls ranged from less than 10% to greater than 300% of Vm. Exchange of residues 357-362 significantly affected both Vm and Km without modifying catalytic efficiency kcat/Km, whereas insertion of residues 346-349 modified Vm and Km and increased catalytic efficiency. Steady state carbanion oxidation rates varied inversely with activity and were differentially affected with respect to equilibrium oxidation rates. Sequence exchange of residues 357-362 appears to modulate carbanion proton exchange, whereas sequence insertion of residues 346-349 modifies substrate and aldehyde interaction with C6 phosphate binding locus. Low intrinsic susceptibility to carboxypeptidase A degradation of the COOH terminus in liver aldolase is consistent with tight association of this COOH terminus in a conformation unfavorable for promoting high catalytic activity. Efficient carbanion protonation promoted by specific sequences 357-362 represents a mechanistic feature which distinguishes catalytically active maize and muscle isozymes from less active liver isozyme. Conservation of active site residues among aldolases suggests that isozyme diversity among aldolases arose from divergent evolution of the COOH-terminal sequence.
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PMID:Differential usage of the carboxyl-terminal region among aldolase isozymes. 849 48

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

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

Molecular imprinting and phage display library technologies are rapidly being accepted as useful techniques for the generation of ligand-selective recognition motifs. The use of molecular imprinting to produce a novel type II aldolase mimic selective for the cobalt(II)-mediated aldol condensation of benzophenone and acetaldehyde is reported here. Furthermore, peptide motifs have been identified which are acting as 'affinity ligands' selective for the recognition of the enzyme alpha-chymotrypsin using phage display techniques.
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PMID:Some recent developments in the preparation of novel recognition systems: a recognition site for the selective catalysis of an aldol condensation using molecular imprinting and specific affinity motifs for alpha-chymotrypsin using a phage display peptide library. 917 52


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