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PubMed2003

The organization of divalent cations in the active site of cadmium Escherichia coli fructose-1,6-bisphosphate aldolase.

Previously determined crystal structures of the zinc enzyme Escherichia coli class II fructose-1,6-bisphosphate aldolase display good agreement for the protein structure but a differing metal-ion organization in the active site. The structure of the enzyme with Cd(2+) in place of Zn(2+) has now been determined to 2.0 A resolution to facilitate cation identification. The protein structure was essentially identical to other structures and five Cd(2+) positions were identified. Two of the cations are at the active site; one corresponds to the catalytic ion and the other provides a structural contribution. These Cd(2+) sites are equivalent to two Zn(2+) ions observed when the enzyme is complexed with a transition-state mimic and confirm our assignment of the roles played by these ions.

Binding Sites↗
PubMed2005

Purification, crystallization and preliminary X-ray crystallographic study of the L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8.

Fuculose phosphate aldolase catalyzes the reversible cleavage of L-fuculose-1-phosphate to dihydroxyacetone phosphate and L-lactaldehyde. The protein from Thermus thermophilus HB8 is a biological tetramer with a subunit molecular weight of 21 591 Da. Purified FucA has been crystallized using sitting-drop vapour-diffusion and microbatch techniques at 293 K. The crystals belong to space group P4, with unit-cell parameters a = b = 100.94, c = 45.87 A. The presence of a dimer of the enzyme in the asymmetric unit was estimated to give a Matthews coefficient (VM) of 2.7 A3 Da(-1) and a solvent content of 54.2%(v/v). Three-wavelength diffraction MAD data were collected to 2.3 A from zinc-containing crystals. Native diffraction data to 1.9 A resolution have been collected using synchrotron radiation at SPring-8.

Aldehyde-Lyases↗
PubMed2002

Chemical-modification rescue assessed by mass spectrometry demonstrates that gamma-thia-lysine yields the same activity as lysine in aldolase.

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.

Amino Acid Sequence↗
PubMed1999

Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications.

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.

Borohydrides↗
PubMed1997

Aldolase C polymorphism in the laboratory opossum, Monodelphis domestica.

A two-allele polymorphism in aldolase C was identified in brain extracts of grey short-tailed opposums, Monodelphis domestica, by electrophoresis in starch or cellulose acetate gels. Pedigree data were consistent with autosomal codominant inheritance. The polymorphism was present in two of the six genetically distinct laboratory populations that have been established for this species.

Alleles↗
PubMed1989

Identification, molecular cloning and sequence analysis of a gene cluster encoding the class II fructose 1,6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli.

To investigate a possible chromosomal clustering of glycolytic enzyme genes, the complete nucleotide sequence of the 8029 bp insert of Escherichia coli DNA in the ColE1 plasmid pLC33-5 of the Clarke and Carbon collection (Clark and Carbon, 1976) was determined. Genes (pgk, fda) encoding the phosphoglycerate kinase and Class II fructose 1,6-bisphosphate aldolase, respectively, of E. coli were identified. The phosphoglycerate kinase was found to be highly homologous in primary structure to the same enzyme from eukaryotic organisms. A further large open reading frame, designated gapB, was also identified, which on the basis of sequence homology, appears to encode another glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase. This putative gene differs significantly from that (designated gapA) already identified as coding for this enzyme in E. coli and which maps elsewhere on the chromosome. The products, if any, of several other open reading frames remain to be identified.

Amino Acid Sequence↗
PubMed2005

Characterization of the molecular mechanisms involved in the differential production of erythrose-4-phosphate dehydrogenase, 3-phosphoglycerate kinase and class II fructose-1,6-bisphosphate aldolase in Escherichia coli.

A gapA-pgk gene tandem coding the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase, is most frequently found in bacteria. However, in Enterobacteriaceae, gapA is replaced by an epd open reading frame (ORF) coding an erythrose-4-phosphate dehydrogenase and an fbaA ORF coding the class II fructose-1,6-bisphosphate aldolase follows pgk. Although epd expression is very low in Escherichia coli, we show that, in the presence of glucose, the 3 epd, pgk and fbaA ORFs are efficiently cotranscribed from promoter epd P0. Conservation of promoter epd P0 is likely due to its important role in modulation of the metabolic flux during glycolysis and gluconeogenesis. As a consequence, we found that the epd translation initiation region and ORF have been adapted in order to limit epd translation and to create an efficient RNase E entry site. We also show that fbaA is cotranscribed with pgk, from promoter epd P0 or an internal pgk P1 promoter of the extended -10 class. The differential expression of pgk and fbaA also depends upon an RNase E segmentation process, leading to individual mRNAs with different stabilities. The secondary structures of the RNA regions containing the RNase E sites were experimentally determined which brings important information on the structural features of RNase E ectopic sites.

Aldehyde Oxidoreductases↗
PubMedDate not supplied

Monoclonal antibody to aldolase C: a selective marker for Purkinje cells in the human cerebellum.

A monoclonal antibody to the glycolytic enzyme aldolase C was used in an immunoperoxidase technique on unfixed and formalin-fixed paraffin-embedded tissues from the human central nervous system (CNS) and other tissues. No staining outside the CNS was detected. In the central nervous system the antibody gave a selective and intense staining of Purkinje cell perikarya, axons and dendrites in both fixed and unfixed tissues. In the human fetal brain, positive staining of Purkinje cells was seen from 35-36 weeks' gestation onwards. This coincides with the conversion of Purkinje cells to a tetraploid state, and may reflect increased glycolytic activity accompanying the rapid dendritic growth at this stage of development. Preliminary investigations on a variety of disease states suggest that this antibody may be useful in studies of ischaemic damage, congenital abnormalities and degenerative conditions affecting the cerebellum.

Adult↗
PubMed1981

Conformational stability of fructose-1, 6-biphosphate aldolase from Ceratitis capitata.

Fructose 1, 6-biphosphate aldolase from Ceratitis capitata is a tetramer of identical subunits with 34% alpha-helix, 22% beta structure and 44% of aperiodic order. Increase of urea concentration up to 4.0 M results in non-cooperative reversible dissociation of the enzyme. Sodium dodecylsulphate 0.06% (w/v) dissociates the tetramer cooperatively with retention of the helical content. Thermal denaturation was a non-reversible cooperative process with a midpoint for the transition at 55 degrees. Cysteine residues are involved in this process and 2-mercaptoethanol preserves partially the enzyme activity. The acidic dissociation of the enzyme is a non-reversible process in contrast to the reversible basic dissociation. Increase of ionic strength results in a more ordered secondary structure for the monomer after acidic dissociation.

Animals↗
PubMed1975

Renaturation of acid-denatured rabbit muscle aldolase. Existence and properties of a stable monomeric intermediate.

The effects of temperature, pH and the substrate, fructose 1,6-bisphosphate, upon the kinetics and yield of renaturation of acid-denatured rabbit muscle aldolase have been investigated. The results are discussed in terms of a sequential set of events leading from the unfolded polypeptide chain to the renatured oligomeric enzyme. One of the intermediate molecular species in this sequence has been characterized as a folded monomer with a sedimentation coefficient of 3.1 S. This monomer is shown to be much more heat-labile than the tetramer under identical conditions, thus demonstrating stabilization of the tertiary structure of the polypeptide chain by the quaternary interactions between protomers.

Animals↗
PubMed1976

Equilibrium studies on the refolding and reactivation of rabbit-muscle aldolase after acid dissociation.

Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH-dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH4-5tetramers and monomers are in equilibrium. Intrinsic chromophores and far-ultraviolet circular dichroism suggest the transition to be a complex multistep process. At pH approximately 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 degrees C, enzyme concentration 0.4-59 mug/ml) up to 95% enzymic activity is recovered which belongs to a renatured tetrameric species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.

Animals↗
PubMed1976

Kinetics of refolding and reactivation of rabbit-muscle aldolase after acid dissociation.

Tetrameric rabbit muscle aldolase has been dissociated to the monomer at pH 2.3 and fully reassociated and reactivated at pH 7.6 Kinetics of reactivation and refolding were followed by slow and fast kinetic techniques after neutralization of the acidic enzyme solutions by dilution or rapid mixing. Rate constants (k), reaction orders (n), and activation evergies (E) were calculated from measurements on the time, concentration, and temperature dependence of the reactions. The experimental results prove reactivation at high enzyme concentration (c greater than 4mug/ml) to obey first-order kinetics; at lower concentrations a transition to a higher reaction order is observed. Because of concentration-dependent deactivation at low enzyme concentrations reactivation measurements could not be extended below c approximately o.5 mug/ml. In the accessible concentration range incomplete separation of reassociation and transconformation processes as well as intrinsic residual activity of the isolated subunits lead to an average value of n = 1.40 +/- 0.18. Renaturation as monitored by protein fluorescnce is a multi-step process composed of a fast increase in flurescence emission (first-order rate constant k approximately 15 s-1) and a slow concentration-dependent decrease which parallels the recovery of enzyme activity (n = 1.46 +/- 0.12). The activation energy of both processes is of the order of E = 12-16 Kcal/mol (50-67 kJ/mol). Reassociation is a prerequisite of full catalytic function and native fluorescence.

Animals↗
PubMed1976

A functional arginine residue in rabbit-muscle aldolase.

Rabbit muscle aldolase is irreversibly modified by the arginine-selective alpha-dicarbonyl, phenylglyoxal, loss of activity correlating with the unique modifications of one arginine residue per subunit, as determined by amino acid analysis, and (7-14C)phenylglyoxal incorporation. The affinity of the modified enzyme for dihydroxyacetone phosphate is significantly reduced while substantial protection against inactivation is afforded by fructose 1,6-disphosphate, dihydroxyacetone phosphate or phosphate ion. The nature of the substrate C-1 phosphate binding site in this enzyme is discussed in the light of these and other results.

Animals↗
PubMed1988

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase.

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.

Animals↗
PubMed1989

The brain-specific gene for rat aldolase C possesses an unusual housekeeping-type promoter.

A DNA fragment encompassing the first exon and about 750 bp of the 5'-flanking sequence has been isolated and sequenced. The gene has multiple start sites of transcription which are dispersed over about 200 bp. The promoter lacks TATA and CAAT boxes and is very G + C-rich, with putative binding sites for the transcriptional factors Sp1 and AP2. Similar features are shared with two other brain-specific genes encoding thy-1 antigen and gamma-enolase. The existence of a conserved block of similarity upstream of the human and rat aldolase C genes suggests that this region could be involved in tissue-specific expression whose mechanism seem to be, at least in part, transcriptional.

Animals↗
PubMed1993

Determinants of the brain-specific expression of the rat aldolase C gene: ex vivo and in vivo analysis.

A 115-bp promoter fragment of the aldolase C gene is sufficient for conferring neural cell specificity on a reporter gene, in cultured PC12 cells and in transgenic mice. In vitro DNase I protection experiments detected two footprints on the promoter, termed boxes A/A', and B. The 5' A/A' box contains overlapping Sp1 and Krox20/Krox24 binding sites; it binds Sp1 in fibroblasts (box A') and a different complex in brain (box A). Any deletion or mutation of this box that impairs protein recognition also suppresses promoter activity. The replacement of box A/A' by a Sp1 consensus binding site results in the loss of the brain specificity of expression in transgenic mice. Further 3', box B is composed of a 5' direct repeat and a 3' GC box consisting of overlapping Sp1 and Krox20/Krox24 binding sites. Mutation of the direct repeat subregion appears to be more deleterious for the promoter activity than mutation of the G+C-rich subregion.

3T3 Cells↗
PubMedDate not supplied

The aldolase and pyruvate kinase isoenzyme patterns in a malignant testicular tumor from a 27 year old male.

On the basis of performed tests, we observed the change of pattern of pyruvate kinase isoenzyme in a malignant testicular tumor (only isoenzyme K) and the same isoenzyme in the tissue of other testis not being tumorously changed. In control group pyruvate kinase isoenzyme K and M was observed. Similarly, another pattern of aldolase isoenzymes in tumorous tissue was stated (AC) comparing with control group (arrangement AC, BC). The changes referred also to the activity of both examined enzymes in tumorous tissue and in the tissue of testis not being tumorously changed. The authors suggest the existence of a factor with depressor characteristics, which is produced by neoplasm and changes the function of genes that are responsible for synthesis examined isoenzymes in the tissue not being tumorously changed.

Adult↗
PubMed1997

Partial purification and characterization of a murine malaria parasite, Plasmodium berghei specific aldolase.

Cell-free P. berghei contains 26.1 times more aldolase activity as compared to normal mouse erythrocytes. Subcellular fractionation of cell-free parasite showed maximum enzyme activity in the soluble fraction. The parasite enzyme was active in a narrow pH range of 7.8-8.0. Of the enzyme activity 90% was lost within 2 weeks at 4 degrees C. Slight inhibition was observed with specific inhibitors ATP, pyrophosphate (PPi) and PEP. The F1, 6DP Km was 0.025 mM.

Animals↗