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)

A simple purification scheme was developed for isolation and purification of cathepsin B from buffalo kidney. The use of CM-Sephadex and chromatofocusing helped in better and simultaneous separation of cathepsin B, H and L. As judged by PAGE and SDS-PAGE studies, the enzyme was found to be pure on the basis of charge and had a molecular mass of 25.5 kDa. The amino acid composition, number of free sulfhydryl groups and other major physico-chemical properties of the purified enzyme were similar to the properties reported for cathepsin B from other sources/tissues. However, the NH2-terminal amino acid residue of the enzyme was found to be Ala as against Leu reported from other tissues/species. The total carbohydrate content was also found to be significantly lower (3.6%) as compared to 7.0-7.6% reported for the enzyme from other sources. Thiol reducing compounds activated the enzyme whereas thiol blocking compounds inhibited it. The buffalo kidney enzyme hydrolyzed Z-Phe-Arg-MCA (Vmax/K(m) = 17.1) as the most efficient substrate followed by Z-Arg-Arg-MCA, BANA and BAPNA. Among the protein substrates, goat hemoglobin (Vmax/K(m) = 874) was found to be the most preferred. Rabbit muscle aldolase, usually considered to be a good substrate for cathepsin B, proved to be a poor substrate for this enzyme; only 25-30% inactivation of aldolase was observed. Antibodies raised against the enzyme recognised only cathepsin B and did not have any cross reactivity with cathepsin H or L from the same or different sources. These differences in the properties of the buffalo kidney enzyme vis-a-vis the same enzyme from other tissue/species have been attributed to specialized function of cathepsin B in diversified tissues.
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PMID:Purification and tissue/species dependence of the specificity of buffalo kidney cathepsin B. 959 26

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.
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PMID:Metabolism of the dimethyl ester of [2,3-(13)C]succinic acid in rat hepatocytes. 987 64

Enzymes proceed the reaction with high regio- and stereoselectivity under mild conditions, i.e. in an aqueous medium at room temperature. However, enzymatic reactions that catalyze carbon-carbon bond formation have not been utilized in organic synthesis until recently. We had an interest in an aldolase-catalyzed reaction which proceed carbon-carbon bond formation referred to aldol condensation, by which many bioactive compounds have been rationally synthesized. On the other hand, recent biological studies on cell recognition (cell adhesion) have disclosed the important roles of oligosaccharides on cell surfaces, especially which include glucuronic acid, 3-deoxy-D-manno-oct-2-ulosonic acid (KDO), and sialic acid in the structures e.g., sialyl Lewis X and endotoxins, in differentiation, induction, viral and bacterial infections, and immune response. As well as acidic oligosaccharides, basic ones have been utilized as practical medicines in the clinical level, like acarbose that acts as an amylase inhibitor. Based on these background, we embarked the synthesis of carbohydrate related compounds which can control the interaction between carbohydrates and carbohydrate recognition protein by the use of several aldolases. Azasugars, potent inhibitors toward glycosidases, were synthesized using fructose-1,6-diphosphate (FDP)-aldolase and other dihdroxyacetonephosphate (DHAP)-dependent aldolases in the key step. Sialyl Lewis X mimetic, peptidic mimetic of RNA having anti-Vero toxin activity, mycestericin D, and aza-idulonic acid were prepared by taking advantage of L-threonine aldolase catalyzed reaction, which afford beta-hydroxy-alpha-L-amino acids. A precursor of KDO, featured acidic sugar of endotoxins was provided by the reaction catalyzed with kynureninase, which generates beta-anion of L-alanine in its active site during the metabolic reaction from kynurenine to anthranilic acid.
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PMID:[Synthesis of carbohydrate related compounds by using aldolase catalyzed reaction]. 1065 81

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.
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PMID:Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases. 1071 19

Muscle actin and fructose-1,6-bisphosphate aldolase (aldolase) were chemically crosslinked to produce an 80 kDa product representing one subunit of aldolase linked to one subunit of actin. Hydroxylamine digestion of the crosslinked product resulted in two 40.5 kDa fragments, one that was aldolase linked to the 12 N-terminal residues of actin. Brownian dynamics simulations of muscle aldolase and GAPDH with F-actin (muscle, yeast, and various mutants) estimated the association free energy. Mutations of residues 1-4 of muscle actin to Ala individually or two in combination of the first four residues reduced the estimated binding free energy. Simulations showed that muscle aldolase binds with the same affinity to the yeast actin as to the double mutated muscle actin; these mutations make the N-terminal of muscle actin identical to yeast, supporting the conclusion that the actin N-terminus participates in binding. Because the depth of free energy wells for yeast and the double mutants is less than for native rabbit actin, the simulations support experimental findings that muscle aldolase and GAPDH have a higher affinity for muscle actin than for yeast actin. Furthermore, Brownian dynamics revealed that the lower affinity of yeast actin for aldolase and GAPDH compared to muscle actin, was directly related to the acidic residues at the N-terminus of actin.
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PMID:Computer simulations of glycolytic enzyme interactions with F-actin. 1108 51

The paper presents the description of Duchenne progressive muscular dystrophy in an 18-month-old and an 8-year-old boy. The diagnosis was established on the basis of clinical symptoms, such as impaired motor development, hypertrophy of leg muscles, difficulty in walking; elevated serum phosphocreatine kinase activity and pathologic electromyographic recordings. The authors emphasize that the disease is characterized by increased activity of such enzymes as: alanine and aspartate aminotransferases, lactate dehydrogenase and aldolase--observed as early as in the first weeks of life, with normal gammaglutamyltranspeptidase activity suggests progressive muscular dystrophy and makes it possible to establish early diagnosis. Early diagnosis of the disease allows to start rehabilitation earlier, to select an appropriate type of anesthesia in case of surgical treatment and to advise parents within the framework of genetic counseling.
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PMID:Early symptoms of Duchenne muscular dystrophy--description of cases of an 18-month-old and an 8-year-old patient. 1120 76

The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.
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PMID:A conserved glutamate residue exhibits multifunctional catalytic roles in D-fructose-1,6-bisphosphate aldolases. 1177 56

The role of active site residues in fructose 1,6-bisphosphate aldolase is investigated by chemical-modification rescue. An active-site mutation, K107C, is constructed in a background where the four solvent-accessible cysteine residues are converted to alanine. The resulting mutant, tetK107C, when reacted with bromoethylamine (BrEA), shows a 40-fold increase in activity (to 80% that of wild type). Determination of the sites and their degree of modification using electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) is developed, allowing correlation of activity after chemical modification rescue to the degree of modification. The stoichiometry of the reaction is 2.5 aminoethylations per subunit, as measured by ESI-FTMS. Protein modification with a double-labeled mix (1:1) of natural abundance isotope (d(0)-BrEA) and 2-bromoethyl-1,1,2,2-d4-amine hydrobromide (d(4)-BrEA), followed by dialysis and trypsin digestion, shows aminoethylated peptides as "twin peptides" separated by four mass units in ESI-FTMS analysis. Using this detection procedure under nondenaturing (native) conditions, C107 is aminoethylated, whereas the four buried thiols remain unlabeled. Aminoethylation of other residues is observed, and correlates with those peptides containing histidine, methionine, and/or the amino terminus. Quantification of the aminoethylation reaction is achieved by labeling with nondeuterated d(0)-BrEA under denaturing conditions following double labeling under native conditions. In addition to complete labeling all five thiols, the intensity of the d(0)-BrEA peak for C107 containing peptides increases, and the change in the d(0)/d(4) ratio between native and denaturing conditions shows 82 +/- 4.5% aminoethylation at C107. This correlation of modification with the recovered activity, indicates that gamma-thia-lysine replaces lysine in the catalytic mechanism. Kinetic constants measured for the rescued K107C mutant enzyme with the substrates fructose 1-phosphate and fructose 1,6-bisphosphate are consistent with the role of the positively charged lysine binding to the C6-phosphate. ESI-FTMS, combined with this double-labeling procedure, allows precise identification of sites and measurement of degree of protein modification.
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PMID:Chemical-modification rescue assessed by mass spectrometry demonstrates that gamma-thia-lysine yields the same activity as lysine in aldolase. 1207 Mar 12

Several studies addressed the question whether needle-EMG causes elevation of muscle enzymes [aspartate-aminotransferase, alanine-aminotransferase, lactate-dehydrogenase, creatine-phosphokinase (CPK), isoenzyme-MB, aldolase] and lactate with conflicting results. However, these studies used sterilizable needle electrodes and different protocols and methods to record EMGs and determine muscle enzymes. This study examined if muscle enzymes are elevated immediately after or 24 h following EMGs with disposable, concentric needle-electrodes, and if they are dependent on age, sex, muscle, number of investigated sites and previous CPK-elevation. In 53 subjects, 24 woman, 29 men, aged 17-88 years, muscle enzymes were determined before, immediately after and 24 h following EMG with disposable, concentric needle-electrodes. Muscle enzymes were not different before, immediately after and 24 h following the EMG. Muscle enzymes were not different between patients <or=60 or >60 years of age. Apart from higher CPK in men than women, muscle enzymes were not different between the genders. Apart from CPK, muscle enzymes were not different between the brachial biceps and anterior tibial muscle. Muscle enzymes were not different if <or=20 or >20 sites were investigated and were independent on pre-EMG CPK-levels. In conclusion this study shows that muscle enzymes do not increase immediately or 24 h following EMG with disposable, concentric needle-electrodes, irrespective of age, gender, muscle, number of investigated sites and pre-EMG CK-levels.
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PMID:Influence of disposable, concentric needle electrodes on muscle enzyme and lactate serum levels. 1212 89

Previous Brownian dynamics (BD) simulations (Ouporov IG, Knull HR and Thomasson KA 1999. Biophys. J. 76: 17-27) of complex formation between rabbit aldolase and F-actin have identified three lysine residues (K288, K293 and K341) on aldolase and acidic residues (DEDE) at the N-terminus of actin as important to binding. BD simulations of computer models of aldolase mutants with any of these lysine residues replaced by alanine show reduced binding energy; the greatest effect of a single substitution is for K341A, and replacement of all three lysines greatly reduces binding. BD simulations of wild-type rabbit aldolase vs altered F-actin show that binding is decreased if any one of the four N-terminal acidic residues is replaced by alanine and binding is greatly reduced if three or more of the N-terminal acidic residues are replaced; none of the four actin residues appear more critical for binding than the others.
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PMID:Brownian dynamics of interactions between aldolase mutants and F-actin. 1250 Nov 61


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