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PubMed1998

Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C.

To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C. Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K. Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli. J Biochem (Tokyo) 1994;115:1172-7). Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K. Isozyme-specific modules on human aldolase A molecule. J Biol Chem 1993;268:1677-83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K. Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence. Prot. Eng. 1994;7:1387-93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C. The kinetic data also suggests that the connector-2 (amino acid residues 243-306) may modulate the interaction of IGS units with the alpha/beta barrel of the aldolase molecule.

Amino Acid Sequence↗
PubMed2001

Quantitative expression studies of aldolase A, B and C genes in developing embryos and adult tissues of Xenopus laevis.

We previously cloned cDNAs for all the members (A, B and C) of Xenopus aldolase gene family, and using in vitro transcribed RNAs as references, performed quantitative studies of the expression of three aldolase mRNAs in embryos and adult tissues. A Xenopus egg contains ca. 60 pg aldolase A mRNA and ca. 45 pg aldolase C mRNA, but contains only ca. 1.5 pg aldolase B mRNA. The percent composition of three aldolase mRNAs (A:B:C) changes from 56:1.5:42.5 (fertilized egg) to 54:10:36 (gastrula), to 71:14.5:14.5 (neurula) and to 73:20:7 (tadpole) during development. These results are compatible with the previous results of zymogram analysis that aldolases A and C are the major aldolases in early embryos, whose development proceeds depending on yolk as the only energy source. Aldolase B mRNA is expressed only late in development in tissues such as pronephros, liver rudiment and proctodeum which are necessary for the future dietary fructose metabolism, and the expression pattern is consistent to that in adult tissues. We also show that three aldolase genes are localized on different chromosomes as single copy genes.

Animals↗
PubMed2002

Phospholipase D2 directly interacts with aldolase via Its PH domain.

Mammalian phospholipase D (PLD) has been implicated in the cellular signal transduction pathways leading to diverse physiological events and known to be regulated by many cellular factors. To identify the proteins that interact with PLD, we performed a protein overlay assay with fractions obtained from the sequential column chromatographic separation of rat brain cytosol using purified PLD2 as a probe. A protein of molecular mass 40 kDa, which was detected by anti-PLD antibody with overlaying of the purified PLD2, is shown to be aldolase C by peptide-mass fingerprinting with matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF-MS). Aldolase A also showed similar binding properties as aldolase C and was co-immunoprecipitated with PLD2 in COS-7 cells overexpressing PLD2 and aldolase A. The PH domain corresponding to amino acids 201-310 of PLD2 was necessary for the interaction observed in vitro, and aldolase A was found to interact with the PH domain of PLD2 specifically, but not with other PH domains. PLD2 activity was inhibited by the presence of purified aldolase A in a dose-dependent manner, and the inhibition by 50% was observed by the addition of less than micromolar aldolase A. Moreover, the inclusion of the aldolase metabolites fructose 1,6-bisphosphate (F-1,6-P) or glyceraldehyde 3-phosphate (G-3-P) resulted in an enhanced interaction between PLD2 and aldolase A with a concomitant increase in the potential ability of aldolase A to inhibit PLD2, which suggests the existence of a possible regulation of the interaction by the change of intracellular concentrations of glycolytic metabolites.

Amino Acid Sequence↗
PubMed1999

Use of FDP and F1P aldolase to detect tumorous and nontumorous tissue damage by ethanol injection of hepatocellular carcinoma.

Changes in serum levels of tumor-specific fructose 1,6-diphosphate (FDP) aldolase and nontumor-specific fructose 1-phosphate (F1P) aldolase activities were analyzed in patients with hepatocellular carcinoma (HCC) to detect the damage of tumorous and nontumorous hepatic cells after percutaneous ethanol injection (PEI). Initial PEI was performed in 20 patients containing 22 HCC nodules with a diameter of < or = 4 cm. Changes in serum hepatic enzyme activities were measured before and after repeated PEI. FDP and F1P aldolase levels were measured by substrate-specific enzymatic methods. Pre- and posttreatment alpha-fetoprotein (AFP) levels were determined by radioimmunoassay. The consequent changes in the total nontumorous liver volumes after PEI were also analyzed by follow-up CT scans. Serum levels of FDP aldolase released by ethanol injection were progressively increased (P < 0.0001) until the third PEI and thereafter decreased. In contrast, serum levels of F1P aldolase were continuously elevated even after the third PEI (P < 0.0001). Serum levels of transaminases were also elevated after repeated PEI (P < 0.0001). The FDP/FIP aldolase ratio decreased significantly with increased volume (>20 ml) of injected ethanol (P = 0.01) caused by nontumorous liver damage. The elevation of FDP aldolase was markedly associated with a decrease in serum levels of AFP (P < 0.001), indicating adequate tumor necrosis. The progression of the total nontumor liver atrophy depended on the volume of injected ethanol and correlated significantly with F1P aldolase levels after PEI (P < 0.01) but not with FDP aldolase. These results demonstrated that caution is needed to avoid nontumorous liver damage caused by the large volume of ethanol injection in treating HCC. Measurement of FDP and F1P aldolase activities in serum after PEI is clinically useful to detect the degree of tumorous and nontumorous tissue damage by ethanol.

Aged↗
PubMed1996

Substrate modulation of aldolase B binding in hepatocytes.

The binding properties of hepatic aldolase (B) were determined in digitonin-permeabilized rat hepatocytes after the cells had been preincubated with either glycolytic or gluconeogenic substrates. In hepatocytes that had been preincubated in medium containing 5 mM glucose as sole carbohydrate substrate, binding of aldolase to the hepatocyte matrix was maximal at low KCl concentrations (20 mM) or bivalent cation concentrations (1 mM Mg2+) and half-maximal dissociation occurred at 50 mM KCl. Preincubation of hepatocytes (for 10-30 min) with glucose or mannose (10-40 mM), fructose, sorbitol, dihydroxyacetone or glycerol (1-10 mM), caused a leftward shift of the salt dissociation curve (maximum binding at 10 mM KCl; half-maximum dissociation at 35 mM KCl) but did not affect the proportion of bound enzyme at low or high KCl concentrations. Galactose and 2-deoxyglucose had no effect on aldolase binding. Inhibitors of glucokinase (mannoheptulose and glucosamine) suppressed the effects of glucose but not the effects of sorbitol, glycerol or dihydroxyacetone. Glucagon suppressed the effects of glucose, fructose and dihydroxyacetone but not glycerol. Poly(ethylene glycol) (PEG) (2-10%), added to the permeabilization medium, increased aldolase binding and caused a rightward shift in the salt dissociation curve. In the presence of PEG (6-8%), the effects of substrates on aldolase dissociation were shifted to higher salt concentrations (50-100 mM versus 35 mM KCl). The effects of substrates (added to the intact cell) on aldolase binding to the permeabilized cell could be mimicked by addition of the phosphorylated derivatives of these substrates to the permeabilized cell. Of the intermediates tested dihydroxyacetone phosphate and fructose 1,6-bisphosphate were the most effective at dissociating aldolase (A50 values of 20 microM and 40 microM respectively). Other effective intermediates in order of decreasing potency were fructose 1-phosphate, glycerol 3-phosphate, glucose 1,6-bisphosphate/fructose 2,6-bisphosphate. These results show that aldolase B binds to the hepatocyte matrix by a salt-dependent mechanism that is influenced by macromolecular crowding and metabolic intermediates. Maximum binding occurs when hepatocytes are incubated in the absence of glycolytic and gluconeogenic substrates and minimum binding occurs in the presence of substrates that are precursors of either fructose 1,6-bisphosphate or triose phosphates. Since the bound form of aldolase represents a kinetically less active state it is proposed that aldolase binding and dissociation may be a mechanism for buffering the concentrations of metabolic intermediates.

Animals↗
PubMed1988

Aldolase exists in both the fluid and solid phases of cytoplasm.

We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 x 10(-7) cm2/S, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 x 10(-7) cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 x 10(-8) cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITC-dextran indicated that FITC-dextran was relatively excluded excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.

Animals↗
PubMed2004

Structure of human brain fructose 1,6-(bis)phosphate aldolase: linking isozyme structure with function.

Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 A resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.

Base Sequence↗
PubMed1996

Functional characterization of an extreme thermophilic class II fructose-1,6-bisphosphate aldolase.

Fructose-1,6-bisphosphate aldolase activity has been isolated and purified to homogeneity from the extreme thermophile eubacteria Thermus aquaticus. The homogeneous enzyme is a class II aldolase as fructose-1,6-bisphosphate cleavage activity was strongly inhibited by EDTA, and activated by Co2+ metal ion. Taq aldolase is a stable tetramer with estimated molecular mass of 165 kDa. The enzyme is thermostable and is not inactived after heating at 90 degrees C for 2 h but looses 80% of activity after 1 h at 97 degrees C. The pH profile corresponding to maximal aldolase activity is displaced to more acidic values compared to other class II aldolases. Enzyme activation by both detergents and alcohols and chromatographic behaviour on hydrophobic stationary phases is consistent with presence of hydrophobic surface regions on the soluble enzyme. Kinetic behaviour of T. aquaticus aldolase at high fructose-1,6-bisphosphate concentrations indicates significant negative cooperativity. The Taq aldolase NH2-terminal sequence was determined and compared with available sequences from other class II aldolases. Significant sequence similarity was found between Taq aldolase and the thermostable aldolase from Bacillus stearothermophilus.

Alcohols↗
PubMed1996

A (G+C)-rich motif in the aldolase C promoter functions as a constitutive transcriptional enhancer element.

The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors.

Animals↗
PubMed1994

Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa.

Two aldolases from the alga Cyanophora paradoxa (Glaucocystophyta) can be separated by chromatography on diethylaminoethyl-Fractogel. The two aldolases are inhibited by 1 mM ethylene-diaminetetraacetate (EDTA) and, therefore, are class II aldolases. When cells of C. paradoxa were fractionated, one aldolase was associated with the cytosol fraction and the other was associated with the cyanoplast fraction. The Km(fructose-1,6-bisphosphate) was 600 [mu]M for the cytosolic aldolase and 340 [mu]M for the cyanoplast aldolase. The activity of the cytosolic aldolase was increased up to 4-fold by 100 mM K+ and slightly inhibited by Li+ and Cs+, whereas the cyanoplast aldolase was not affected by these ions. Inactivation by 1 mM EDTA could be partly restored by the addition of Co2+ or Mn2+ and to a lesser extent by Zn2+ or Mg2+. The molecular masses of the native cytosolic and cyanoplast aldolases are about 90 and 85 kD, respectively, as estimated by velocity centrifugation in sucrose gradients. Implications for the evolution of class I and II aldolases in chloroplasts of higher plants and algae will be discussed.

Journal Article↗
PubMed2001

Starvation-induced lysosomal degradation of aldolase B requires glutamine 111 in a signal sequence for chaperone-mediated transport.

Aldolase B is an abundant cytosolic protein found in all eukaryotic cells. Like many glycolytic enzymes, this protein was sequestered into lysosomes for degradation during nutrient starvation. We report here that the degradation of recombinant aldolase B was enhanced two-fold when rat and human hepatoma cells were starved for amino acid and serum. In addition, starvation-induced degradation of aldolase B was inhibited by chloroquine, an inhibitor of lysosomal proteinases and by 3-methyladenine, an inhibitor of autophagy. Aldolase B has three lysosomal targeting motifs (Q(12)KKEL, Q(58)FREL, and IKLDQ(111)) that have been proposed to interact with hsc73 thereby initiating its transport into lysosomes. In this study, we have mutated the essential glutamine residues in each of these hsc73-binding motifs in order to evaluate their roles in the lysosomal degradation of aldolase B during starvation. We have found that when glutamines 12 or 58 are mutated to asparagines enhanced degradation of aldolase B proceeded normally. However, when glutamine 111 was mutated to an asparagine or a threonine, starvation-induced degradation was completely suppressed. These mutations did not appear to alter the tertiary structure of aldolase B since enzymatic activity was not affected. Our results suggest that starvation-induced lysosomal degradation of aldolase B requires both autophagy and glutamine 111. We discuss the possible roles for autophagy and hsc73-mediated transport in the lysosomal sequestration of aldolase B.

Amino Acid Motifs↗
PubMed1990

Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation.

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.

Amino Acids↗
PubMed1981

Proteolytic modification of rat liver fructose-1,6-bisphosphate aldolase by administration of leupeptin in vivo.

When leupeptin, a thiol protease inhibitor of microbial origin, was injected into rats, the activity of fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) in the liver decreased to about 60% of that in control rats. However, the concentration of aldolase protein in the liver extracts, measured with a specific antibody obtained with enzyme purified on a phosphocellulose column, remained unchanged. Injection of leupeptin also caused a marked increase in the activities of free lysosomal proteases, such as cathepsin B (EC 3.4.22.1), cathepsin L (EC 3.4.22.-), cathepsin D (EC 3.4.23.5) and lysosomal carboxypeptidase A in the cytosol fraction. A clear inverse relationship between aldolase and cathepsin B activities in the cytosol fraction was demonstrated. The possibility that the less active form of aldolase detected in the livers of leupeptin-treated rats was produced during homogenization was excluded by showing that the aldolase activity was not changed by addition of various protease inhibitors to the homogenization medium., When insulin was coinjected with leupeptin, increase in the activity of free cathepsin L and decrease of activity of aldolase produced by the injection of leupeptin was prevented. These findings indicate that modification of aldolase may be due to the action of a lysosomal protease(s). Enhanced sensitivity of lysosomes to osmotic shock was demonstrated in the livers of leupeptin-treated rats, suggesting that the lysosomal membrane is labilized by administration of leupeptin. Incubation of the purified aldolase with the lysosomal fraction produced the same changes in properties of aldolase as those observed in vivo on injection of leupeptin.

Animals↗
PubMed1992

Aldolase-DNA interactions in a SEWA cell system.

In this report we demonstrate the novel finding that aldolase A interacts with DNA sequences in mouse SEWA sarcoma cells. This interaction was initially observed through the identification of a 40 kDa protein which was eluted from a DNA affinity chromatography column consisting of the long terminal repeat (LTR) of the endogenous intracisternal A-type particle (IAP). Microsequencing analysis identified this 40 kDa protein as the glycolytic enzyme, aldolase A. The use of specific anti-aldolase antibodies enabled the identification and subsequent purification of aldolase from the nuclear protein fraction of two SEWA sublines, one that is adherent and one that grows in suspension. In order to confirm our initial finding that aldolase is capable of interacting with DNA, proteins from each subline were immunopurified with anti-aldolase antibodies, eluted and then tested for their ability to interact with IAP-LTR DNA sequences. Interestingly, only aldolase derived from the anchorage dependent SEWA cells was capable of interacting with the IAP-LTR, however, several cell lines derived from human tumors also exhibited this activity. Subsequent studies revealed the ability of aldolase to interact with some but not every DNA sequence tested, implying that there may be a minimal DNA conformation and/or sequence requirement for this activity. The presence of aldolase A in the nuclei and its ability to interact with certain DNA sequences suggest a novel role for this metabolic enzyme.

Animals↗
PubMed2004

The interaction of FBPase with aldolase: a kinetic and fluorescence investigation on chicken muscle enzymes.

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 microM Fru-1,6P2 and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with Ks approximately 5 microM and Ksi approximately 39 microM and factor beta approximately 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (Kd) of 3.8 microM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase interaction results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The Kd of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 microM at 25 degrees C with almost 1:1 stoichiometry. This interaction increases the I(0.5) for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the Ka and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase.

Adenosine Monophosphate↗
PubMed1996

The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold.

BACKGROUND: [corrected] Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution. The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Amino Acid Sequence↗
PubMed1995

Solution structure of a band 3 peptide inhibitor bound to aldolase: a proposed mechanism for regulating binding by tyrosine phosphorylation.

Human erythrocyte band 3 inhibits glycolytic enzymes, including aldolase, by binding these cytoplasmic enzymes at its N-terminus. Phosphorylation of Y8 disrupts inhibition, and there is evidence that in vivo glycolysis levels in erythrocytes are regulated in part by a phosphorylation/dephosphorylation signaling pathway. The structural basis for control by phosphorylation has been investigated by NMR studies on a complex between aldolase and a synthetic peptide corresponding to the first 15 residues of band 3 (MEELQDDYEDMMEEN-NH2). The structure of this band 3 peptide (B3P) when it is bound to rabbit muscle aldolase was determined using the exchange-transferred nuclear Overhauser effect (ETNOE). Two hundred NMR structures for B3P were generated by simulated annealing molecular dynamics with NMR-derived distance restraints and excluding electrostatic terms. Twenty structures were further refined against a force field including full partial charges. The important conformational feature of B3P in the bound state is a folded loop structure involving residues 4-9 and M12 that surrounds Y8 and is stabilized by a hydrophobic cluster with the ring of Y8 sandwiched between the methyl groups of L4 and M12. Differential line broadening indicates that this loop structure binds aldolase in a relatively specific manner, while terminal regions are structurally heterogeneous. To better understand B3P inhibition of aldolase and the mechanism of phosphorylation control, a complex was modeled by docking B3P into the active site of aldolase and optimizing the fit using restrained molecular dynamics and energy minimization. The B3P loop is complementary in conformation to the beta-barrel central core containing the aldolase active site residues. Binding is electrostatic in nature with numerous ionic and hydrogen-bonding interactions involving several conserved lysine and arginine residues of aldolase. How phosphorylation of band 3 could disrupt inhibition was considered by modeling a phosphoryl moiety onto Y8 of B3P. An energetic analysis with respect to rigid phosphate rotation suggests that aldolase inhibition is reversed primarily because of electrostatic repulsion between B3P residues that destabilizes the B3P loop formed in the complex. This proposed intramolecular mechanism for blocking protein--protein association by electrostatic repulsion with the phosphoryl group may be applicable to other protein--protein signaling complexes.

Amino Acid Sequence↗
PubMed1973

A comparative study of aldolase from human muscle and liver.

Aldolase was purified from human skeletal muscle and human liver by techniques capable of processing large quantities (10-20kg) of tissue. The methods used also proved convenient for isolating aldolase on a large scale from other mammalian and avian sources. Aldolase from both human liver and muscle was crystallized; each gave two crystalline forms, depending on the conditions of crystallization. X-ray studies on the muscle aldolase crystals suggest a close structural similarity between human and rabbit muscle aldolase. Aldolases from human muscle and liver have similar pH optima and pH stability but their stability to heat treatment differs. The effect of heat on the enzymes may therefore provide an easy means of distinguishing them. The kinetic constants K(m) and k(cat.) for these aldolases are similar to other mammalian aldolases. Amino acid analyses and tryptic peptide ;mapping' show that the primary structures of the two aldolases differ greatly.

Amino Acids↗