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)

2-Keto-4-hydroxyglutarate aldolase, which catalyzes the reversible cleavage of 2-keto-4-hydroxyglutarate, yielding pyruvate plus glyoxylate, has been purified from extracts of bovine kidney to apparent homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration chromatography, sucrose density gradient centrifugation, and meniscus depletion sedimentation equilibrium experiments. The enzyme from this source has a native and a subunit mass of 144 and 36 kDa, respectively; the pH-activity optimum is 8.8. Rather than being stimulated, aldolase activity is inhibited to varying degrees by added divalent metal ions, whereas a number of metal ion-chelating agents have no effect. An absolute requirement for added thiol compounds could not be shown, but 2-mercaptoethanol enhances activity 2-fold, and added Hg2+ as well as p-mercuribenzoate or dithiodipyridine markedly inhibit catalysis. Incubation of the enzyme with either pyruvate or glyoxylate in the presence of NaBH4 causes extensive loss of aldolase activity concomitant with stable binding of approximately 1.0-1.5 mol of 14C-labeled substrate/mol of enzyme. The circular dichroism spectrum for native aldolase is characteristic of an alpha-helix; incubation of the enzyme with glyoxylate has no effect on this spectrum, but it is considerably altered by pyruvate. Bovine kidney aldolase shows no stereospecificity in catalyzing the aldol cleavage of the two optical isomers of 2-keto-4-hydroxyglutarate, and although it also catalyzes the beta-decarboxylation of oxalacetate, its decarboxylase/aldolase activity ratio is lower than that seen with the pure enzyme from either bovine liver or Escherichia coli.
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PMID:2-Keto-4-hydroxyglutarate aldolase: purification and characterization of the homogeneous enzyme from bovine kidney. 158 31

Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.
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PMID:Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase. 309 43

Treatment of homogeneous preparations of Escherichia coli 2-keto-4-hydroxyglutarate aldolase with 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal, or 2,4-pentanedione results in a time- and concentration-dependent loss of enzymatic activity; the kinetics of inactivation are pseudo-first order. Cyclohexanedione is the most effective modifier; a plot of log (1000/t 1/2) versus log [cyclohexanedione] gives a straight line with slope = 1.1, indicating that one molecule of modifier reacts with each active unit of enzyme. The kinetics of inactivation are first order with respect to cyclohexanedione, suggesting that the loss of activity is due to modification of 1 arginine residue/subunit. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced structural alteration of the aldolase. The same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. Amino acid analyses of 95% inactivated aldolase show the loss of 1 arginine/subunit with no significant change in other amino acid residues. Considerable protection against inactivation is provided by the substrates 2-keto-4-hydroxyglutarate and pyruvate (75 and 50%, respectively) and to a lesser extent (40 and 35%, respectively) by analogs like 2-keto-4-hydroxybutyrate and 2-keto-3-deoxyarabonate. In contrast, formaldehyde or glycolaldehyde (analogs of glyoxylate) under similar conditions show no protective effect. These results indicate that an arginine residue is required for E. coli 2-keto-4-hydroxyglutarate aldolase activity; it most likely participates in the active site of the enzyme by interacting with the carboxylate anion of the pyruvate-forming moiety of 2-keto-4-hydroxyglutarate.
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PMID:Evidence for an essential arginine residue in the active site of Escherichia coli 2-keto-4-hydroxyglutarate aldolase. Modification with 1,2-cyclohexanedione. 388 56

The possibility is examined that 4-hydroxy-2-ketoglutarate aldolase (4-hydroxy-2-ketoglutarate glyoxylatelyase, EC 4.1.3.16), the last step in hydroxyproline catabolism is regulated by intermediates of gluconeogenesis. Inhibition of isolated 4-hydoxy-2-ketoglutarate aldolase was examined using dual inhibition studies. It was found that the enzyme exhibits synergistic inhibition by oxaloacetate and pyruvate, but only when the substrate concentration is low. At substrate concentrations approaching saturation, the inhibition by the oxaloacetate and pyruvate becomes additive. These results are discussed in terms of possible control of the use of carbon from hydroxyproline breakdown in glucose production.
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PMID:Regulation of rat liver 4-hydroxy-2-ketoglutarate aldolase. 394 59

The enzyme 4-hydroxy-2-ketoglutarate aldolase (4HKG aldolase), which catalyzes the reversible cleavage of 4-hydroxy-2-ketoglutarate to form pyruvate and glyoxylate, was isolated from rat liver. The purification scheme as well as a study of several of the physical and kinetic properties of the enzyme are presented. The effects of anions, various buffers, and possible physiologically relevant effectors on the kinetic parameters of the aldolase were also investigated. It was found that pyruvate analogs inhibited the aldolase. Oxaloacetate was a competitive inhibitor of the aldolase, and in addition caused synergistic inhibition with respect to pyruvate analogs at low substrate concentration. These results are discussed in terms of possible regulation of the aldolase.
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PMID:Rat liver 4-hydroxy-2-ketoglutarate aldolase: purification and kinetic characterization. 396 4

The alpha-ketoglutarate dehydrogenase complex of either pig heart or Escherichia coli catalyzes a NAD- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate which is stereoselective toward the L-isomer of this hydroxyketo acid. L-Malyl-CoA is the product of the reaction; the evidence includes observing (a) a steady increase in absorbance at 230 nm during the oxidation of 2-keto-4-hydroxyglutarate, (b) a positive response of oxidation reaction mixtures to neutral hydroxylamine, (c) loss of the two foregoing results concomitant with release of thiol-reacting material and the formation of free malate when reaction mixtures are heated, (d) formation of a hydroxamate which has chromatographic mobilities identical to that of chemically synthesized malate hydroxamate, (e) enzymatic formation of a radioactive product from 14C-labeled 2-keto-4-hydroxyglutarate which co-migrates with chemically synthesized malyl-CoA, and (f) hydrolysis of the product by citrate synthase, an enzyme absolutely specific for citryl-CoA and L-malyl-CoA. A 1:1:1 stoichiometric relationship exists between the amount of 2-keto-4-hydroxyglutarate oxidized, NAD reduced, and malate (or malyl-CoA) formed. Results from studies in which either 14C-labeled 2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate is incubated with mixtures of purified enzymes or extracts of E. coli support the suggestion that the aldolase which preferentially catalyzes formation of L-2-keto-4-hydroxyglutarate from pyruvate plus glyoxylate in E. coli is coupled with the oxidative decarboxylation of this substrate, as reported here, and other enzymes in a multistep pyruvate-catalyzed cyclic oxidation of glyoxylate.
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PMID:Malyl-CoA formation in the NAD-, CoASH-, and alpha-ketoglutarate dehydrogenase-dependent oxidation of 2-keto-4-hydroxyglutarate. Possible coupled role of this reaction with 2-keto-4-hydroxyglutarate aldolase activity in a pyruvate-catalyzed cyclic oxidation of glyoxylate. 638 79

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

Perturbations in glyoxylate metabolism lead to the accumulation of oxalate and give rise to primary hyperoxalurias, recessive disorders characterized by kidney stone disease. Loss-of-function mutations in HOGA1 (formerly DHDPSL) are responsible for primary hyperoxaluria type III. HOGA1 is a mitochondrial 4-hydroxy-2-oxoglutarate aldolase catalyzing the fourth step in the hydroxyproline pathway. We investigated hydroxyproline metabolites in the urine of patients with primary hyperoxaluria type III using gas chromatography-mass spectroscopy. Significant increases in concentrations of 4-hydroxy-2-oxoglutarate and its precursor and derivative 4-hydroxyglutamate and 2,4-dihydroxyglutarate, respectively, were found in all patients as compared to carriers of the corresponding mutations or healthy controls. Despite a functional block in the conversion of hydroxyproline to glyoxylate--the immediate precursor of oxalate--the production of oxalate increases. To explain this apparent contradiction, we propose a model of glyoxylate compartmentalization in which cellular glyoxylate is normally prevented from contact with the cytosol where it can be oxidized to oxalate. We propose that HOGA1 deficiency results in the accumulation of 4-hydroxy-2-oxoglutarate in the mitochondria and its transport into the cytosol where it is converted to glyoxylate by a different cytosolic aldolase. In human hepatocyte cell lines, we detected a cytosolic 4-hydroxy-2-oxoglutarate aldolase activity not due to HOGA1. These studies provide a diagnostic tool for primary hyperoxaluria type III and shed light on glyoxylate metabolism and the pathogenesis of primary hyperoxalurias.
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PMID:Primary hyperoxaluria type III--a model for studying perturbations in glyoxylate metabolism. 2272 92

Carboligations catalyzed by aldolases or thiamine diphosphate (ThDP)-dependent enzymes are well-known in biocatalysis to deliver enantioselective chain elongation reactions. A pyruvate-dependent aldolase (2-oxo-3-deoxy-6-phosphogluconate aldolase [EDA]) introduces a chiral center when reacting with the electrophile, glyoxylic acid, delivering the (S)-enantiomer of (4S)-4-hydroxy-2-oxoglutarate [(S)-HOG]. The ThDP-dependent enzyme MenD (2-succinyl-5-enol-pyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (SEPHCHC synthase)) enables access to highly functionalized substances by forming intermolecular C-C bonds with Michael acceptor compounds by a Stetter-like 1,4- or a benzoin-condensation 1,2-addition of activated succinyl semialdehyde (ThDP adduct formed by decarboxylation of 2-oxoglutarate). MenD-catalyzed reactions are characterized by high chemo- and regioselectivity. Here, we report (S)-HOG, in situ formed by EDA, to serve as new donor substrate for MenD in 1,4-addition reactions with 2,3-trans-CHD (2,3-trans-dihydroxy-cyclohexadiene carboxylate) and acrylic acid. Likewise, (S)-HOG serves as donor in 1,2-additions with aromatic (benzaldehyde) and aliphatic (hexanal) aldehydes. This enzyme cascade of two subsequent C-C bond formations (EDA aldolase and a ThDP-dependent carboligase, MenD) generates two new stereocenters.
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PMID:Extended substrate range of thiamine diphosphate-dependent MenD enzyme by coupling of two C-C-bonding reactions. 3006 80