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)

Conditions were determined in which approximately one mole of omicron-phthalaldehyde reacts with one mole of aldolase subunit yielding a stable fluorescent isoindole derivative. During this chemical modification, a linear relationship was observed between the enzyme inactivation and absorbance change (337 nm) or fluorescence change (lambda em 420 nm, and lambda ex 338 nm) characteristic for isoindole ring formation. The reaction follows second-order kinetics, k = 1.1 X 10(3) M-1 S-1, in 50 mM borate buffer, pH 8.4 at 25 degrees C. The modification of aldolase results in loss of approximately one -SH group per protein subunit. The enzyme is protected against modification by substrates and competitive inhibitors. Essentially no isoindole derivative is formed when the glycerol-1-phosphate-lysyl derivative of aldolase is used for modification studies. It is concluded that aldolase modification occurs at the active-site region. Isolation of cross-linked peptides suggests that Lys-227 and Cys-336 are involved in formation of the isoindole derivative. This result supports Cys-336 as the active-site cysteine necessary for aldolase catalytic activity. Fluorescence studies have shown that the isoindole group linked to aldolase has its lambda max, em markedly shifted toward shorter wavelength in comparison to the fluorescence of free isoindole derivatives in aqueous solution. In model studies a linear relationship between lambda max, em of 1-(beta-hydroxyethylthio)-2-beta-hydroxyethylisoindole and the solvent polarity or acidity was observed. The results of the studies suggest that the microenvironment of the cleft in aldolase which binds isoindole appears to be of low acidity and low polarity. The apparent low polarity experienced by the isoindole probe may be due to its location in an actual low-polarity portion of the active site, or may be due to non-relaxing surroundings of the probe.
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PMID:o-Phthalaldehyde, a fluorescence probe of aldolase active site. 666 5

Some experimental and clinical studies were done from the metabolic viewpoint to elucidate the characteristics of myonephropathic-metabolic syndrome. In experimental dogs with their femoral arteries ligated and two third of femoral muscles divided, aldolase and myoglobin showed remarkable increase without significant changes in electrolytes. Slight increase of GPT and GOT was observed. Amino acids showed elevation in urea, taurin, leucin, isoleucin, valine, threonine, 3-methylhistidine, phenylalanine, histidine, lysine, methionine, tyrosine and anserin and decrease in glutamine, alanine, glycine, proline, carnosine, citrullin and arginine. In patients with acute arterial occlusion, potassium, GOT, LDH, CPK, lactate and pyruvate increased moderately and myoglobin showed remarkable increase and aldolase slight increase. Amino acids showed remarkable increase in 3-methylhistidine and beta-amino-isobutyric acid and moderate increase in phenylalanine and arginine. These results revealed that measurement of free amino acid concentration, especially that of methylhistidine as well as myoglobin, pyruvate, lactate and some other enzymes might be of great help to predict the prognosis of patients with acute arterial occlusion of the extremities.
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PMID:[Metabolic study on acute arterial occlusion of the extremities]. 667 89

Alkanediol monoglycolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P), which are analogues of the aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) substrate fructose 1,6-bisphosphate, were synthesized and used for probing its active site. The Ki value was lowest when the maximum distance between the phosphorus atoms of the bisphosphate was brought close to that of fructose 1,6-bisphosphate. The binding constants estimated from difference spectra correlate well with Ki values for the substrate analogues. Propanediol monoglycolate bisphosphoric ester protected aldolase from inactivation by 1,2-cyclohexanedione, which preferentially attacks arginine-55. However, propanol phosphate had little protective effect. The synthesized phosphate compounds protected the enzyme against inactivation by trypsin, and also against spontaneous denaturation. These results suggest that the synthesized phosphate compounds bind to aldolase at the active site, which tends to keep the distance constant between the two phosphate-binding sites for the open-chain form of fructose 1,6-bisphosphate, and stabilize the natural conformation of the enzyme. Both arginine-55 and lysine-146 are shown to participate in the phosphate-binding site for the C-1-phosphate of fructose 1,6-bisphosphate.
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PMID:An exploration of the binding site of aldolase using alkanediol monoglycolate bisphosphoric esters. 682 94

The COOH-terminal BrCN fragment of the aldolase alpha-subunit from muscles of rabbits in norm and under atherosclerosis was studied by the method of dansyl-fingerprints in a silicagel and polyamide thin layer. It is shown that under atherosclerosis the amount of peptides in the fragment under study increases and the topography of two of them changes. The content of lysine, serine and valine enhances in it. The results evidence for structural differences in C-terminal fragment of aldolase alpha-subunits in muscles of rabbits in norm and under experimental atherosclerosis.
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PMID:[Structural differences of C-terminal fragment of the alpha-subunits of rabbit muscle aldolase in normal animals and in experimental atherosclerosis]. 713 8

Under atherosclerosis the fractions corresponding to alpha-subunits are focused at a more alkaline pH than the same fractions in the norm. The curve of the enzymic activity of the fractions with atherosclerosis is higher. beta-subunits of aldolase from muscles of intact rabbits and those with sclerosis are identical in the amino acidic composition. In the enzyme alpha-subunits under conditions of atherosclerosis the content of lysine, serine, glycine, valine gets higher. On the basis of the previous research which reveals peptide having no analogs in the norm in the C-terminal fragment of aldolase molecule an assumption is advanced that under conditions of atherosclerosis the intermediate C-terminal site of the enzyme alpha-chain changes.
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PMID:[Amino acid composition and subunit structure of rabbit muscle aldolase in experimental atherosclerosis]. 721 Feb 25

S-Carboxymethylated chicken muscle aldolase was treated with cyanogen bromide to cleave the 4 methionyl bonds per subunit. Five homogeneous fractions were obtained designated fragments I-V. Fragment I was derived from the N-terminus and fragment II from the C-terminus of the enzyme. Reduction of the enzyme with NaB3H4 in the presence of dihydroxyacetone phosphate decreases the enzymatic activity by 90%. Fragment III contained the Schiff base-forming lysine residue since more than 83% of the radioactivity introduced by NaB3H4 reduction of aldolase-dihydroxyacetone phosphate was found in this fraction. A tryptic peptide of 27 amino acid residues containing the substrate-binding site was isolated. The gross molecular structure of aldolase A from chicken muscle indicates a high degree of homology with mammalian muscle aldolases.
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PMID:Studies on the structure of aldolase A from chicken muscle. 721 8

A Ni(2+)-binding protein (pNiXc, 40 kDa), present in Xenopus laevis oocytes and embryos, was isolated from mature oocytes by chromatography on DEAE-cellulose and cellulose phosphate, followed by FPLC on Ni-iminodiacetate-Agarose, or reverse-phase HPLC on a C-4 column. Size-exclusion HPLC showed that intact pNiXc is approximately 155 kDa, consistent with tetrameric structure. After cleavage with Lys-C proteinase or cyanogen bromide, six peptides were separated by HPLC and sequenced by Edman degradation, providing sequence data for 83 residues. Data-base search showed similarity of pNiXc to eukaryotic aldolases, with 96% identity to human aldolase A. pNiXc demonstrated aldolase activity with fructose 1,6-bisphosphate as substrate (Km, 30 microM Vmax 26 mumol min-1 mg-1); the aldolase activity was inhibited non-competitively by Cu2+, Cd2+, Co2+, or Ni2+. Equilibrium dialysis showed high affinity binding (Kd, 7 microM) of 1 mole of Ni per mole of 40 kDa subunit. Based on metal-blot competition assays, the abilities of metals to compete with 63Ni2+ for binding to pNiXc were ranked: Cu2+ >> Zn2+ > Cd2+ > Co2+. This study identifies pNiXc as the monomer of fructose-1,6-bisphosphate aldolase A, and raises the possibility that aldolase A is a target enzyme for metal toxicity.
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PMID:The 40 kDa 63Ni(2+)-binding protein (pNiXc) on western blots of Xenopus laevis oocytes and embryos is the monomer of fructose-1,6-bisphosphate aldolase A. 787 95

Lysine-146 of rabbit muscle aldolase (D-fructose-1,6-biphosphate aldolase, EC 4.1.2.13) is absolutely conserved in class I (Schiff base) aldolases and has been implicated previously in catalysis by protein modification. Site-directed mutagenesis was used to change lysine-146 to alanine, glutamine, leucine, or histidine, creating the mutant enzymes K146A, K146Q, K146L, and K146H, respectively. These mutant proteins were expressed at high levels in bacteria and were purified by substrate affinity elution from CM-Sepharose, the same method that is used for the wild-type enzyme. The mutants K146A, K146Q, and K146L had substrate cleavage rates below standard detection levels. Modified cleavage assays indicated that these enzymes were (0.5-2) x 10(6)-fold decreased in the rate of catalysis of fructose 1,6-bis(phosphate) (Fru-1,6-Pa)cleavage. The K146H enzyme, however, was approximately 2000-fold slower than wild type in the rates of both cleavage and condensation of Fru-1,6-P2. In assays for the presence of enzymatic intermediates, all of the mutant enzymes were able to catalyze formation of the carbanion intermediate with dihydroxyacetone phosphate, whereas this intermediate was below the level of detection with Fru-1,6-P2. Single-turnover experiments with these enzymes in excess over radiolabeled Fru-1,6-P2 were used to measure the rates of Schiff base and product formation. The rate of Schiff base formation was decreased in each of the mutant enzymes, yet the magnitude of this decrease was less than the reduction in the respective kcat. These mutations had a much larger effect, however, on the rate of C3-C4 bond breaking, showing that Lys-146 is crucial at this step of the catalytic cycle.
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PMID:Lysine-146 of rabbit muscle aldolase is essential for cleavage and condensation of the C3-C4 bond of fructose 1,6-bis(phosphate). 791 50

The expression and purification of the rabbit muscle aldolase A (D-fructose 1,6-bisphosphate:D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from an expression plasmid in bacteria is described. The enzyme is produced in bacteria at a level of 300 mg/liter and is indistinguishable from the enzyme isolated from muscle in assays using fructose 1,6-bisphosphate and fructose 1-phosphate. The recombinant enzyme has the same primary, secondary, and quaternary structure as the muscle enzyme. Aspartic acid 33, found near the active site lysine in the crystal structure, is changed to alanine, serine, and glutamic acid by site-directed mutagenesis, resulting in the mutant proteins, D33A, D33S, and D33E, respectively. The mutant enzymes are purified by substrate affinity elution from carboxylmethyl-Sepharose, the same method as that used for the wild-type enzyme. The secondary and quaternary structure of D33A is identical to wild-type aldolase when analyzed by light scattering, gel filtration, and circular dichroism. Moreover, the hexose substrate can be fixed in the active site by reduction of the Schiff base with sodium borohydride, indicating that the active site is not drastically altered. These single mutations in the active site have a serious effect on the activity of the enzyme. In addition, the rate of carbanion oxidation for D33A is 17-29 times slower when the substrate is fructose 1,6-bisphosphate versus dihydroxyacetone phosphate, whereas in the wild-type there is no significant difference in these rates. This evidence and the conservation of this residue in other class I aldolases indicate that aspartic acid 33 is an essential residue in the catalytic mechanism, possibly involved in abstraction of the carbon 4 hydroxyl proton.
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PMID:Site-directed mutagenesis identifies aspartate 33 as a previously unidentified critical residue in the catalytic mechanism of rabbit aldolase A. 841 16

Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527-2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse-phase HPLC. Mass spectroscopy, 1H- and 13C-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of the two lysine residues: [formula: see text]
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PMID:Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site. 851 1


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