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)

A gel penetration technique, that measures the dilution undergone by protein equilibrium on a short tightly packed gel column, has been employed to determine the molecular masses of aldolase (160 kDa), glyceraldehyde-3-phosphate dehydrogenase (GPDH; 145 kDa) in the absence and presence of each other and of other proteins. The dilution factor (concentration of protein applied/concentration of protein after equilibration) was found to be inversely related to the molecular mass of the protein. In equimolar mixtures of aldolase and GPDH, 0.5-2.5 microM each, the two enzymes exhibited a common molecular mass value of 309-316 kDa. These enzymes did not undergo any self association or disassociation in this concentration range. Moreover, their molecular masses were unaffected by the presence of other proteins tested. When the concentration of one of these enzymes (aldolase or GPDH) was held constant and that of the other varied, the dilution factor of the former was decreased as the concentration of the latter was increased until it corresponded to a molecular mass of ca. 310 kDa at equimolar concentrations of the two enzymes. Further increase in the concentration of the variable enzyme had no effect. It has been suggested that aldolase and GPDH form a 1:1 complex of dissociation constant equal to or less than 5 x 10(-8) M. The complex was found to dissociate in the presence of KCl, (NH4)2SO4, ATP and NADH whereas its formation was favoured by fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, NAD+, ADP, AMP and phosphate ions.
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PMID:Interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase: molecular mass studies. 924 8

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD+, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca2+/calmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission.
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PMID:The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide. 937 36

A protein-protein association regulated by phosphorylation of tyrosine is examined by NMR structural studies and biochemical studies. Binding of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and aldolase to the N-terminus of human erythrocyte anion transporter, band 3, inhibits enzyme activity. This inhibition is reversed upon phosphorylation of band 3 Y8, as shown by kinetic studies on purified components, as well as in vivo studies. Thus, tyrosine phosphorylation mediates against the intermolecular protein-protein association, in contrast to the positive control involving SH2 and PTB domains where phosphorylation is required for binding. To elucidate the basis of recognition and negative control by tyrosine phosphorylation, the structure of a synthetic peptide, B3P, corresponding to the first 15 residues of band 3 (MEELQDDYEDMMEEN-NH2), bound to G3PDH has been determined using the exchange-transferred nuclear Overhauser effect. The G3PDH-bound B3P structure was found to be very similar to the structure recognized by aldolase. A hydrophobic triad forms from side chains within a loop structure of residues 4 through 9 in both bound species. Another structural feature stabilizing the loop, in the case of the B3P-G3PDH complex, is a hydrogen bond between the side chains of Y8 and D10 associated with a beta-turn of residues 8-11. Based on the structure of this phosphorylation sensitive interaction (PSI) loop, it is suggested that tyrosine phosphorylation disrupts protein-protein association, in part, by intramolecular electrostatic destabilization. The inhibition by B3P is competitive with respect to the coenzyme NAD+ and noncompetitive with the substrate analog arsenate. Specific binding of B3P to G3PDH is demonstrated by reversion of the NMR spectral properties of bound B3P to those of the free peptide upon addition of coenzyme and substrate analog. The stoichiometry of binding for the B3P-G3PDH complex was determined from Sephadex G-50 displacement experiments to be 4:1. Collectively, these results are consistent with B3P binding the active site of G3PDH.
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PMID:Insights into tyrosine phosphorylation control of protein-protein association from the NMR structure of a band 3 peptide inhibitor bound to glyceraldehyde-3-phosphate dehydrogenase. 945 76

Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.
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PMID:Epimerization via carbon-carbon bond cleavage. L-ribulose-5-phosphate 4-epimerase as a masked class II aldolase. 954 61

A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate--fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 +/- 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the curvette concentration range of 0.1 microM to 0.1 mM.
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PMID:A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues. 976 43

Intrinsic chemical properties of the zinc(II) ion in zinc enzymes have been investigated by the model of 1:1 Zn2+-macrocyclic polyamine complexes, including Zn2+-1,5,9-triazacyclododecane ([12]aneN3) and 1,4,7,10-tetraazacyclododecane (cyclen). The physiologically most suitable pKa values for the Zn2+-bound H2O in enzymes were illustrated by the first model Zn2+-[12]aneN3 complex, which mimics the essential kinetic and thermodynamic roles of Zn2+ in carbonic anhydrase. The activation of proximate serine residues (in alkaline phosphatase) and activation of alcohols for hydride transfer to NAD+ (in alcohol dehydrogenase) were also mimicked by Zn2+ -[12]aneN3 complexes. The functions of two zincs in dinuclear metallophosphatases were explained by a new dinuclear Zn2+-cryptate. For an aldolase type II model, a Zn2+-cyclen derivative showed facile enolate formation from a proximate carbonyl pendant under physiological conditions. The strong anion affinities, which Zn2+ intrinsically possesses, were exploited into novel selective nucleobase thymine (or uracil) recognition of Zn2+-cyclen complexes by the strong Zn2+ -imido anion bond formation. The Zn2+-aromatic-pendant cyclen complexes selectively bind to T (or U) in single- and double-stranded DNA (or RNA). Thus, Zn2+ complexes act like molecular zippers to break A-T pairs in DNA, which was proven by various physicochemical measurements and DNA footprinting assays. These Zn2+ complexes showed some relevant biochemical and biological properties such as inhibition of transcriptional factor, TATA binding protein, or strong antimicrobial activities to gram-positive bacterial strains.
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PMID:Why zinc in zinc enzymes? From biological roles to DNA base-selective recognition. 1081 60

The compartmentation of key processes in sugar, organic acid and amino acid metabolism was studied during the development of the flesh and seeds of grape (Vitis vinifera L.) berries. Antibodies specific for enzymes involved in sugar (cell wall and vacuolar invertases, pyrophosphate: fructose 6-phosphate phosphotransferase, aldolase, NADP-glyceraldehyde-P dehydrogenase, cytosolic fructose 1,6-bisphosphatase), photosynthesis (Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase), amino acid metabolism (cytosolic and mitochondrial aspartate aminotransferases, alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase), organic acid metabolism (phosphoenolpyruvate carboxylase, NAD- and NADP-dependent malic enzyme, ascorbate peroxidase), and lipid metabolism (acetyl CoA carboxylase, isocitrate lyase) were used to determine how their abundance changed during development. There were marked changes in the abundance of many of these enzymes in both the flesh and seeds. The intercellular location of some enzymes was investigated using immunohistochemistry. Several enzymes (e.g. phosphoenolpyruvate carboxylase and those involved in amino acid metabolism) were associated with tissues likely to function in the transport of imported assimilates, such as the vasculature. Although other enzymes (e.g. NADP-malic enzyme and soluble acid invertase, involved in the metabolism of sugars and organic acids) were largely present in the parenchyma cells of the flesh, their distribution was extremely heterogeneous. This study shows that when considering the metabolism of complex structures such as fruit, it is essential to consider how metabolism is compartmentalized between and within different tissues, even when they are apparently structurally homogeneous.
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PMID:An immunohistochemical study of the compartmentation of metabolism during the development of grape (Vitis vinifera L.) berries. 1093 59

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.
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PMID:Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase. 1101 22

The final two steps of the meta-cleavage pathway for catechol degradation in Pseudomonas sp. strain CF600 involve the conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A by the enzymes 4-hydroxy-2-ketovalerate aldolase and NAD(+)-dependent acylating aldehyde dehydrogenase. Biochemical studies indicate that these two enzymes comprise a bifunctional heterodimer (DmpFG, molecular mass 71 kDa) and suggest that the product of the aldolase reaction is transferred to the dehydrogenase active site via a channeling mechanism. Crystals of the DmpFG complex grow in multiple fan-like clusters of thin plates by the hanging-drop method and are improved by streak-seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 102.0, b = 140.7, c = 191.3 A, and diffract to 2.1 A resolution. The asymmetric unit contains four DmpFG heterodimers. Heavy-atom derivative screening identified three isomorphous derivatives.
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PMID:Crystallization and preliminary X-ray analysis of dmpFG-encoded 4-hydroxy-2-ketovalerate aldolase--aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600. 1126 89

Aspergillus niger could utilize D-galactose as sole source of carbon. Cell-free extracts of D-galactose-grown mycelia were able to catalyze the oxidation of D-galactose to D-galactonic acid-gamma-lactone (GalA-gamma-lact) in the presence of NAD, followed by the appearance of 2-keto-3-deoxy-D-galactonate (KDGal), pyruvate and glyceraldehyde. From 10 &mgr;moles only 6.6 &mgr;moles of GalA-gamma-lact were disappeared after 60 min of reaction indicating the presence of GalA-gamma-lactonase. Identification of GalA-gamma-lact was achieved by ascending paper chromatography. KDGal, pyruvate and glyceraldehyde were also chromatographically identified in the reaction mixture containing D-galactonate which suggests that D-galactonate is degraded into pyruvate and glyceraldehyde via the intermediate formation of KDGal. Such reactions are supposed to be catalyzed by an inducible D-galactonate dehydratase and a constitutive KDGal aldolase. The amount of KDGal, pyruvate and glyceraldehyde were found to be almost equivalent and the equilibrium of the reaction being toward the formation of KDGal. The apparent equilibrium constant (K(eq)) was calculated and found to be 0.5 x 10(-3) M. Results also proved the reversibility of the reaction catalyzed by KDGal aldolase of A. niger. In the light of the findings obtained from the degradation of D-galactose by cell-free extracts of A. niger grown on D-galactose and D-galactonate a nonphosphorolytic pathway was suggested to be operative for the degradation of D-galactose in extracts of A. niger.
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PMID:Evidence for a non-phosphorylated route of galactose breakdown in cell-free extracts of Aspergillus niger. 1142 38


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