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)

After a brief exposition to glucose, Thiobacillus acidophilus was isolated from a culture of iron-grown T. ferrooxidans. Physicochemical analysis of its DNA showed a G+C content of 62.9-63.2%. The new isolate grows best at 25-30 degrees C and at pH 3.0. Growth is possible between pH 1.5 and 6.0. Thiobacillus acidophilus is apparently strictly aerobic. Ammonium salts are the only suitable source of nitrogen. The bacterium is a facultative autotroph. In addition to elemental sulfur, it obtains energy from organic compounds such as D-glucose, D-galactose, D-fructose, D-mannitol, D-xylose, D-ribose, D-arabinose, L-arabinose, sucrose, sodium citrate, malic acid,dl-aspartic acid, and dl-glutamic acid. Thiobacillus acidophilus possesses the key enzymes of the tricarboxylic acid (TCA) cycle including NAD-and NADP-linked isocitric dehydrogenase and alpha-ketoglutarate dehydrogenase, and the key enzymes of the hexose monophosphate pathway (glucose-6-phosphate and 6-phosphogluconate dehydrogenase, and fructose 1,6-diphosphate aldolase). NADH oxidase has been found in particulate fraction of extracts. Rhodanese and thiosulfate oxidase have also been detected.
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PMID:Thiobacillus acidophilus sp. nov.; isolation and some physiological characteristics. 23 84

Enzymatic studies on aldolase isozymes have been carried out by techniques of protein engineering. Site-directed mutagenesis helps us to verify the roles of amino acid residues in catalytic reactions. Chimeric fusion proteins give us information about the regions which specify the characteristics of the isozymes. The results are: (1) In aldolase A, COOH terminal Tyr and Lys-107 residues play important roles in catalysis, especially in binding of FDP. (2) Aspartic acid at the 128th residue in aldolase A is essential to thermostability; no other residue such as glutamic acid can substitute for it. (3) Studies on chimeric fusion proteins indicate that the C-terminal region (including C-terminus Tyr) or aldolase A is responsible for its substrate specificity, which is not seen in aldolase B. (4) A region near NH2 terminus in aldolase B determines its specific structure. (5) The region including His-107, Asp-128, and Tyr-137 (B-A junction of BA137) is located in a turn which is exposed outward (a model architecture by Sygusch et al [1987]). In BA137, this region would be constrained, and play a significant role in catalysis, thermostability, etc. (6) Tertiary structure of aldolase B seems to be dissimilar to that of aldolase A.
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PMID:Structural studies on aldolase isozymes through protein engineering. 220 67

Fructose-1,6-bisphosphate aldolase A (fructose-bisphosphate aldolase; EC 4.1.2.13) deficiency is an autosomal recessive disorder associated with hereditary hemolytic anemia. To clarify the molecular mechanism of the deficiency at the nucleotide level, we have cloned aldolase A cDNA from a patient's poly(A)+ RNA that was expressed in cultured lymphoblastoid cells. Nucleotide analysis of the patient's aldolase A cDNA showed a substitution of a single nucleotide (adenine to guanine) at position 386 in a coding region. As a result, the 128th amino acid, aspartic acid, was replaced with glycine (GAT to GGT). Furthermore, change of the second letter of the aspartic acid codon extinguished a F ok I restriction site (GGATG to GGGTG). Southern blot analysis of the genomic DNA showed the patient carried a homozygous mutation inherited from his parents. When compared with normal human aldolase A, the patient's enzyme from erythrocytes and from cultured lymphoblastoid cells was found to be highly thermolabile, suggesting that this mutation causes a functional defect of the enzyme. To further examine this possibility, the thermal stability of aldolase A of the patient and of a normal control, expressed in Escherichia coli using expression plasmids, was determined. The results of E. coli expression of the mutated aldolase A enzyme confirmed the thermolabile nature of the abnormal enzyme. The Asp-128 is conserved in aldolase A, B, and C of eukaryotes, including an insect, Drosophila, suggesting that the Asp-128 of the aldolase A protein is likely to be an amino acid residue with a crucial role in maintaining the correct spatial structure or in performing the catalytic function of the enzyme.
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PMID:Human aldolase A deficiency associated with a hemolytic anemia: thermolabile aldolase due to a single base mutation. 282 99

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

Cathepsin B was isolated from buffalo liver by salt fractionation, ion-exchange resin treatment, gel filtration and repeated ion-exchange chromatography using a linear salt gradient. The enzyme showed activity, against denatured hemoglobin (or ovalbumin), alpha-N-benzoyl-DL-arginine p-nitroanilide (BAPNA), and alpha-benzoyl-DL-arginine-naphthylamine (BANA). It inactivated buffalo muscle aldolase with a half life period of 21 min. The pH-activity profiles obtained for the digestion of hemoglobin (or ovalbumin) and aldolase inactivation by the enzyme were found to be different. The enzyme (mol wt 27,800 by SDS-PAGE) eluted in gel filtration with a molecular weight of 27,000 and a Stokes radius of 2.31 nm. The results showed buffalo cathepsin B to be a single-chain molecule. The N- and C-terminal amino acids of the enzyme were found to be leucine and aspartic acid, respectively. It contained 0.7% concanavalin A reactive neutral carbohydrate. The amino acid composition of buffalo cathepsin B was found to be similar to that of human liver cathepsin B. The optical properties of the buffalo enzyme were found consistent with its aromatic amino acid content. The isoionic pH of the enzyme was found to be 5.70 and the intrinsic viscosity was 3.48 ml/g whence the frictional ratio, f/f0 was computed to be 1.10 suggesting that the native enzyme conformation is compact and is globular in solution.
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PMID:Isolation, purification and properties of cathepsin B from buffalo liver. 893 19

Malaria remains a major disease of mankind, and resistance to existing therapeutics is rapidly emerging. Limited financial investment to develop new therapeutics requires the careful selection of well-defined targets from the causative parasite, Plasmodium falciparum. In these circumstances, protein crystallography can provide valuable structural detail to facilitate both the selection of suitable targets and the development of compounds to provide novel drug candidates. This review summarises the current involvement of crystallographic studies in anti-malarial drug development programmes. Protein crystallography is increasingly central to the exploitation of a number of potential Plasmodial targets. including the aspartic acid proteases (plasmepsins) and cysteine proteases (falcipains) involved in haem degradation within the parasite food vacuole. Lead compounds are being identified from collections previously synthesised against homologous human enzymes. Plasmodium have an unusual dependence on the glycolytic pathway relative to their human hosts, and this is reflected in subtle structural differences identified in the crystal structures of a number of parasite glycolytic enzymes including aldolase and lactate dehydrogenase. Other enzymes from a range of biosynthetic pathways have also been targeted in crystallographic studies. These include dihydrofolate reductase, the target of existing anti-folate therapeutics, and enoyl reductase from the fatty acid biosynthesis pathway which is already the target of effective bacteriocides. Crystal structures of these drug-enzyme complexes not only allow visualisation and improvement of inhibitor-protein contacts, but in the former case have also been used to probe the molecular basis of emerging anti-malarial drug resistance. Crystallography is similarly proving valuable as a tool to facilitate the development of inhibitors of purine salvage, isoprenoid synthesis and utilisation, and protein processing mechanisms.
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PMID:Structure-based approaches to the development of novel anti-malarials. 1501 47

The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine. Using active site mutants of FBPA I from Thermoproteus tenax, we have solved the crystal structures of the enzyme covalently bound to the carbinolamine of the substrate fructose 1,6-bisphosphate and noncovalently bound to the cyclic form of the substrate. The structures, determined at a resolution of 1.9 A and refined to crystallographic R factors of 0.148 and 0.149, respectively, represent the first view of any FBPA I in these two stages of the reaction pathway and allow detailed analysis of the roles of active site residues in catalysis. The active site geometry of the Tyr146Phe FBPA variant with the carbinolamine intermediate supports the notion that in the archaeal FBPA I Tyr146 is the proton donor catalyzing the conversion between the carbinolamine and Schiff base. Our structural analysis furthermore indicates that Glu187 is the proton donor in the eukaryotic FBPA I, whereas an aspartic acid, conserved in all FBPA I enzymes, is in a perfect position to be the general base facilitating carbon-carbon cleavage. The crystal structure of the Trp144Glu, Tyr146Phe double-mutant substrate complex represents the first example where the cyclic form of beta-fructose 1,6-bisphosphate is noncovalently bound to FBPA I. The structure thus allows for the first time the catalytic mechanism of ring opening to be unraveled.
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PMID:Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates. 1576 50

Monoclonal antibody (mAb) 38C2 belongs to a group of catalytic antibodies that were generated by reactive immunization and contains a reactive lysine. 38C2 catalyzes aldol and retro-aldol reactions, using an enamine mechanism, and mechanistically mimics natural aldolase enzymes. In addition, mAb 38C2 can be redirected to target integrins alpha(v)beta(3) and alpha(v)beta(5) through the formation of a covalent bond between a beta-diketone derivative of an arginine-glycine-aspartic acid (RGD) peptidomimetic and the reactive lysine residue in the antibody combining site to provide the chemically programmed mAb cp38C2. In this study, we investigated the potential of enhancing the activity of receptor-binding small molecule drug (SCS-873) through antibody conjugation. Using a M21 human melanoma xenograft model in nude mice, cp38C2 inhibited the growth of the tumor by 81%. The chemically programmed antibody was shown to be highly active at a low concentration while SCS-873 alone was ineffective even at dosages 1,000-fold higher than those used for the chemically programmed antibody. In vitro programming of the catalytic antibody was shown to be as effective as in vivo programming. In an experimental metastasis assay, treatment with mAb cp38C2 significantly prolonged overall survival of tumor-bearing severe combined immuno-deficient (SCID) mice when compared to treatment with unprogrammed mAb 38C2, SCS-873 alone or the integrin-specific monoclonal antibody LM609. In vitro, cp38C2 inhibited human and mouse endothelial and human melanoma cell adhesion, migration and invasion. Additionally, cp38C2 inhibited human and mouse endothelial cell proliferation and was active in complement-dependent cytotoxicity assays. These studies establish the potential of chemically programmed monoclonal antibodies as a novel and effective class of immunotherapeutics that combine the merits of traditional small molecule drug design with immunotherapy.
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PMID:Small molecule drug activity in melanoma models may be dramatically enhanced with an antibody effector. 1657 Feb 83

Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA) is a member of the class II zinc-dependent aldolase family that catalyzes the cleavage of d-fructose 1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced with an alanine residue renders the enzyme inactive. A comparison of the crystal structures of D83A FBPA in complex with FBP and of wild-type FBPA in the unbound state revealed a substrate-induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn(2+). When FBP binds, the Zn(2+) shifts up to 4.6 A toward the catalytic Asp83, which brings the metal within coordination distance of the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor d-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analogue of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA-FBP structure. The mechanism invokes coordination and coplanarity of the Zn(2+) with the FBP's O-C(3)-C(4)-O group concomitant with coordination of the Asp83 carboxylic group. Catalysis is accompanied by movement of Zn(2+) to a site coplanar with the O-C(2)-C(3)-O group of the DHAP. glFBPA exhibits strict substrate specificity toward FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP versus TBP specificity. Replacement of Asp255 with an alanine resulted in an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; k(cat)/K(m) = 80 M(-1) s(-1)) while maintaining activity toward FBP at a 50-fold lower catalytic efficacy compared with that of wild-type FBPA. The collection of structures and sequence analyses highlighted additional residues that may be involved in substrate discrimination.
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PMID:Structural insights into the substrate binding and stereoselectivity of giardia fructose-1,6-bisphosphate aldolase. 1923 2

Ciauscolo is a short-ripened fermented sausage manufactured in the Marche region (central Italy) that has recently received a protected geographical indication product classification (PGI). The aim of this study was the exploration of the biochemical traits of this traditional Italian salami, with a special focus on protein and lipid composition. Ciauscolo salami was characterized by pH of 5.1 and 0.91 water activity. A prevalence of lactic acid bacteria in the microbiota was found. The free amino acids and biogenic amines average content was 2657 and 255 mg/kg, respectively. With regards to lipids composition unsaturated fatty acids represented 63% and 72% of total and free fatty acids. Despite these results had wide statistical variability, attributable to differences in the processing parameters and raw matter used, some peculiar traits were found: (1) structural muscular proteins underwent to less proteolysis than sarcoplasmic ones; (2) glycogen phosphorylase, enolase, and aldolase were the most proteolyzed among the sacoplasmic proteins; (3) there was inverse correlation between histamine content and yeasts population, and a direct correlation between the gly-pro content and lactic acid bacteria counts; (4) the content of aspartic acid and methyonine seem to be a possible molecular marker able to distinguish between double and single milling.
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PMID:Biochemical traits of Ciauscolo, a spreadable typical Italian dry-cured sausage. 2072 5


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