Mammalian Aldolases Research Articles

Comparisons of Phosphofructokinases-1 from Rabbit, Chicken, and Fish

Background: Previous studies suggested that glycogen storage was facilitated by ascorbate inhibition of phosphofructokinase-1 (PFK-1) in resting mammalian muscle; these studies showed that purified PFK-1 from fish or chicken muscle has properties similar to PFK-1 from mammalian muscles. Materials and Methods: The enzymes utilized in the assay systems came from Sigma-Aldrich Co. Rabbit (Oryctolagus cuniculus) muscle G-actin (A 2522) was free of aldolase, LDH (EC 1.1.1.28) or AK (EC 2.7.4.3) activity and rabbit muscle aldolase (EC 4.1.2.13) was free of AK (EC 2.7.4.3) and LDH activity. Rabbit muscle PFK-1 (RPFK-1), chicken (Gallus gallus) muscle PFK- 1 (CPFK-1) and Pacific red snapper (Lutjanus peru) muscle PFK-1 (FPFK-1) used in these tests were prepared from frozen tissues with modifications of a method. AK, LDH, and aldolase activity were absent in purified FPFK- 1 and CPFK-1 preparations. Results: It can be shown that the following enzymes associated with glycolysis are not inhibited by 0.1 M ascorbate under our conditions: rabbit muscle aldolase (EC 4.1.2.13); rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12); rabbit muscle phosphoglucose isomerase (EC 5.3.1.9); rabbit muscle pyruvate kinase (EC 2.7.1.40); yeast hexokinase (EC 2,7.1.1); and yeast 3-phosphoglyceric phosphokinase (EC 2.7.2.3). Rabbit muscle enolase (EC4.2.1.11) is inhibited under our conditions. Conclusion: In summary, FPFK-1 and CPFK-1, possess characteristics and behaviors similar to RPFK-1, e.g., losses of activities due to dilutions and protections of some of these activity losses by rabbit muscle aldolase. These interactions of a mammalian aldolase with fish and a bird PFK-1’s suggests a conservative evolutionary relationship among aldolases and PFK-1’s.

Bisphosphonate Inhibitors of Mammalian Glycolytic Aldolase.

The glycolytic enzyme aldolase is an emerging drug target in diseases such as cancer and protozoan infections which are dependent on a hyperglycolytic phenotype to synthesize adenosine 5'-triphosphate and metabolic precursors for biomass production. To date, structural information for the enzyme in complex with phosphate-derived inhibitors has been lacking. Thus, we determined the crystal structure of mammalian aldolase in complex with naphthalene 2,6-bisphosphate (1) that served as a template for the design of bisphosphonate-based inhibitors, namely, 2-phosphate-naphthalene 6-bisphosphonate (2), 2-naphthol 6-bisphosphonate (3), and 1-phosphate-benzene 4-bisphosphonate (4). All inhibitors targeted the active site, and the most promising lead, 2, exhibited slow-binding inhibition with an overall inhibition constant of ∼38 nM. Compound 2 inhibited proliferation of HeLa cancer cells, whereas HEK293 cells expressing a normal phenotype were not inhibited. The crystal structures delineated the essential features of high-affinity phosphate-derived inhibitors and provide a template for the development of inhibitors with prophylaxis potential.

Identification of leishmania fructose-1,6-bisphosphate aldolase as a novel activator of host macrophage Src homology 2 domain containing protein tyrosine phosphatase SHP-1

The macrophage protein tyrosine phosphatase-1 SHP-1 has been implicated in the pathogenesis of infection with leishmania. To identify the factors that may interact with SHP-1, Leishmania donovani promastigote lysates were added to a GST-SHP-1 affinity matrix. A 44kDa specifically bound protein was identified as leishmania fructose-1,6-bisphosphate aldolase (aldolase). Purified leishmania aldolase bound to SHP-1 indicating that the interaction was direct. In contrast, purified mammalian aldolase did not bind to SHP-1. Consistent with this, leishmania aldolase activated SHP-1 in vitro, whereas mammalian aldolase did not. The presence of leishmania aldolase in the cytosolic fractions prepared from infected macrophages indicated that leishmania aldolase is exported from phagolysosomes in infected cells where it can target host cytosolic proteins. In fact, co-immunoprecipitation showed association of leishmania aldolase with SHP-1. Moreover, leishmania aldolase-expressing macrophages showed the deactivated phenotype of leishmania infected cells as judged by much reduced inability to induce expression of nitric-oxide synthase in response to interferon-γ treatment. Collectively, these data show that leishmania aldolase is a novel SHP-1 binding and activating protein that contributes to macrophage dysfunction.

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys(146) --> Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys(229) in native aldolase. Mutation of conserved active site residue Lys(146) to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr(363) hydroxyl within 2.9A of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr(363) is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys(146) positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr(363) interaction with Lys(146) and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.

Modeling the interaction between aldolase and the thrombospondin-related anonymous protein, a key connection of the malaria parasite invasion machinery

A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 A) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds.

Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes

Sialate pyruvate-lyases, also known as sialate aldolases (EC 4.1.3.3), reversibly catalyse the cleavage of free N-acetylneuraminic acids to form pyruvate and N-acetylmannosamine. These enzymes are widely distributed and are present in numerous pro- and eukaryotic cells, in which they are localized only in the cytosol. They play an important role in the regulation of sialic acid metabolism by controlling the intracellular concentration of sialic acids of biosynthetic or exogenous origin, thus preventing the accumulation of toxic levels of this sugar. Application of an original colorimetric micromethod for N-acetylmannosamine determination, as well as the use of [4,5,6,7,8,9- 14C] N-acetylneuraminic acid, led us to evidence a cytosolic neuraminate aldolase activity in human red blood cells (RBCs) and then to define the main characteristics of this enzyme: Michaelis–Menten type, K m: 1.4 ± 0.05 mM, optimal pH: 7.6 ± 0.2, optimal temperature: 70 ± 2 °C, inhibition by heavy metals: Ag + and Hg ++. These enzyme parameters are close to those of the bacterial and mammalian aldolases described up to now. At the moment, the presence of sialate pyruvate-lyase in the cytosol of red blood cells remains an enigma.

Structures of Type 2 Peroxisomal Targeting Signals in Two Trypanosomatid Aldolases

Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These “PTS2 dimers”, which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.

Structure of aldolase A (muscle-type) cDNA and its regulated expression in oocytes, embryos and adult tissues of Xenopus laevis

We obtained cDNA (XALDA; 1466 bp) for Xenopus laevis aldolase A gene (muscle-type), whose amino acid sequence had 88% similarity to those of mammalian aldolase A genes. XALDA mRNA occurred abundantly in skeletal muscle and at low levels also in other adult tissues, and such mRNA distribution was reflected in zymograms. In oocytes XALDA mRNA occurred at a relatively high level from stage I, and the mRNA level peaked at stage II, then decreased in later stages. XALDA mRNA in the full-grown oocyte was inherited as maternal mRNA throughout maturation and fertilization until midblastula stage, but its level became very low during gastrula and early neurula stages, and then increased greatly in later stages. While maternal XALDA mRNA was distributed uniformly in early embryos, mRNA zygotically expressed after late neurula stage occurred mainly in somites. In blastula animal caps XALDA mRNA occurred at a low level, but the expression was greatly enhanced by activin treatment. Thus, in Xenopus laevis aldolase A gene is actively transcribed in earlier phase of oogenesis, inherited as maternal mRNA in early embryos in a cell-type nonspecific way, then in later phases of embryogenesis, it is strongly expressed in somites with its concomitant ubiquitous expression at low levels in almost all the other cell types.

Specificity and inhibitory activity of antibodies to Plasmodium falciparum aldolase.

The multiplication of Plasmodium falciparum within RBC is energy-dependent and the glucose consumption of infected RBC is increased more than 50 times over the consumption of normal RBC. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase (p41) have been detected in infected RBC. Expression of the cloned aldolase gene of P. falciparum in Escherichia coli resulted in an enzymatically active polypeptide with a high sp. act. and the recombinant p41 aldolase was used for enzymatic and immunologic studies reported here. The presence of antibodies against p41 in the sera of human adults partially immune to malaria and immunization experiments in monkeys suggest that p41 is implicated in protective immune response against the parasite. Therefore, we analyzed the capacity of various antisera to inhibit P. falciparum aldolase activity. It was found that anti-p41 antibodies raised in mice, rabbits, and monkeys inhibited very efficiently aldolase activity in vitro up to dilutions higher than 10(-3). In contrast none of the human sera with high levels of anti-p41 antibodies were able to inhibit parasite aldolase activity even at a dilution of 1/2. The inability of human antisera to neutralize parasite aldolase is not related to antibody titers but is probably related to the specificity of the human antibodies. This finding is discussed in relation to homology of structure of P. falciparum and mammalian aldolase and to a possible mechanism of parasite adaptation and survival in its natural host.

Amino acid sequence of an invertebrate FBP aldolase (from Drosophila melanogaster)

The complete amino acid sequence of FBP aldolase from Drosophila melanogaster has been determined. The enzyme contains four identical subunits of 360 amino acid residues. The primary structure of the monomer was established using automated Edman degradation on fragments prepared by CNBr-cleavage, by partial acid cleavage at the unique Asp-Pro bond and by oxidative cleavage at the three tryptophan residues. Manual Edman-Chang degradation was used on smaller peptides obtained by digestion with Staphylococcus aureus V8 protease, trypsin or chymotrypsin. The primary structure of Drosophila aldolase exhibits very extensive homology with the sequence of rabbit muscle aldolase (71% identity), thus explaining the early observation that Drosophila and mammalian aldolases form active interspecies hybrid quaternary structures (Brenner-Holzach, O. and Leuthardt, F., Eur. J. Biochem. (1972) 31 , 423–426).

38] Fructose-bisphosphate aldolase from human erythrocytes

Publisher Summary This chapter describes an assay for fructose-bisphosphate aldolase from human erythrocytes that is based on the oxidation of NADH by dihydroxyacetone phosphate in a coupled system containing triosephosphate isomerase and α-glycerophosphate dehydrogenase. The purification process involves washing of erythrocytes and hemolysis, batch process, column chromatography, and reverse ammonium sulfate fractionation. Isoelectric focusing reveals the presence of one form of aldolase in the erythrocyte with an isoelectric point of 8.9. Sedimentation equilibrium ultracentrifugation and gel filtration chromatography reveals a native molecular weight of 158,000. The enzyme, like other mammalian aldolases, appears to be a tetramer with identical or near identical subunits. The amino acid composition is very similar to that of the human heart and rabbit muscle enzymes. The human erythrocyte aldolase appears to be a type A isozyme or muscle-type aldolase.

Fructose-bisphosphate aldolase from Helix pomatia—II. Chemical and physical properties

1. 1. Fructose-bisphosphate aldolase from Helix pomatia is a tetramer of 40,000 mol. wt. sub-units like mammalian aldolases. 2. 2. The snail enzyme differs slightly in amino acid composition from mammalian aldolases and has glycine as its amino terminus rather than proline. 3. 3. Spectroscopic measurements (u.v., fluorescence, ORD, CD) show small yet definite differences in secondary structure between the snail and mammalian aldolases but indicate thaT no major structural changes have occurred during evolution.

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.

Structural Comparisons between the Class I Fructose Diphosphate Aldolases from Micrococcus aerogenes and Rabbit

It has been previously shown that the fructose-P2 aldolase isolated from Micrococcus aerogenes is a Class I (Schiff base) enzyme. All other prokaryotic organisms so far studied contain Class II (metallo) aldolases. Studies were undertaken to investigate whether or not this bacterial aldolase is structurally related to the Class I aldolases of higher organisms. 1. A comparison of amino acid compositions gives no indication of homology between M. aerogenes and mammalian aldolases. 2. The NH2-terminal residue of this enzyme was found to be methionine in contrast to proline which is found at the NH2 terminus of the rabbit enzymes. 3. The M. aerogenes enzyme contains lysine rather than tyrosine at the COOH terminus. 4. The tryptic peptide containing the active site lysyl residue of M. aerogenes aldolase was isolated and partially characterized. The peptide contains 28 amino acids as do the peptides of rabbit aldolases A and B. The amino acids of the M. aerogenes peptide may be aligned to obtain the maximum of 48% homology with the corresponding rabbit aldolase peptides. 5. Several of the structural distinctions between M. aerogenes and rabbit aldolases are discussed in relation to the proposed functions of tyrosine, and cysteine in the catalytic process of Class I fructose-P2 aldolases. Our results suggest extensive and perhaps fundamental differences between the M. aerogenes enzyme and the Class I aldolases of mammals. The extent of relationship between this prokaryotic enzyme and the fructose-P2 aldolases of higher organisms must await more thorough structural analyses.

Purification and properties of fructose diphosphate aldolase from ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K m values of ascarid aldolase are 1 × 10 −6 m and 2 × 10 −3 m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH 4 in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.

Heterogeneity of Drosophila aldolase and hybridization with mammalian aldolase

Abstract–1. FDP aldolase from pupae of Drosophila melanogaster is shown by the following findings to be a tetrameric molecule with two non‐identical subunits: (a) Disc electrophoresis on polyacrylamide gel in the presence of sodium dodecylsulfate yields a subunit molecular weight of 40000 (equal to that of rabbit muscle aldolase). (b) Hybridisations of drosophila aldolase with type C aldolase from rabbit and calf brain lead to a five‐membered set of isoenzymes.2. The non‐identity of the subunits is suggested by the behaviour on isoelectro focussing. While rabbit muscle aldolase and human liver aldolase show five isoenzymes (hybrids from the non‐identical subunits), drosophila aldolase can be separated into three peaks only. This result may be interpreted in two ways: (a) The isoenzymes α4 and α3α′ are not separated from each other in the pH range 5‐‐8, neither are αα′3 and α′4. Or (b) α2 and α′2 are not split under the condition of isoelectro focussing and therefore 3 isoenzymes α4, α2α′2, and α′4 are obtained only.(3) Isoenzyme I (supposedly mainly α4) and isoenzyme III (supposedly mainly α′4) are identical in the following respects: Michaelis constant, pH profile, FDP/F‐1‐P activity ratio, inactivation by carboxypeptidase A, disc electrophoresis on polaycrylamide and molecular weight. The only difference found between isoenzyme I and III is the specific activity: Isoenzyme I is nearly twice as active as isoenzyme III.4. The fact that hybridisation between drosophila aldolase and type C aldolase from rabbit and calf brain yields a set of isoenzymes proves that interspecies hybridisation is possible between FDP aldolases from vertebrates and invertebrates. FDP aldolases from vertebrates and invertebrates seem to be homologous enzymes. Our results suggest that the conformation of aldolases remained relatively unchanged during evolution.

Lobster muscle aldolase: Isolation, properties and primary structure at the substrate-binding site

Fructose 1,6- P 2 aldolase has been isolated from lobster muscle. The crystalline enzyme is shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis and gel filtration. The enzyme has a molecular weight of 160,000 based on sedimentation equilibrium, sedimentation velocity and gel filtration. The amino acid composition of the lobster muscle aldolase is similar to that of rabbit muscle aldolase, except for the content of half-cystine, methionine and aromatic amino acids. Tyrosine is found to be the COOH-terminal amino acid by carboxypeptidase A digestion and hydrazinolysis. The release of 4 moles of tyrosine per mole of enzyme by carboxypeptidase A digestion suggests a tetrameric structure of lobster muscle aldolase similar to that of other mammalian aldolases. The kinetic parameters are similar to those described for rabbit muscle aldolase, except that lobster muscle aldolase has a much lower rate of cleavage of fructose 1- P as compared with fructose 1,6- P 2. The amino acid sequence of the active site peptide isolated from tryptic digests has been determined. It shows extensive homology with similar peptides isolated from vertebrate muscle and liver. A region containing 12 amino acids immediately adjacent to the lysine residue which forms the Schiff base intermediate is identical in all aldolases thus far examined.

Comparative studies on muscle aldolases purified from rat, rabbit, human, pigeon, and hen.

Muscle aldolases [EC 4.1. 2.13] have been purified from rat, rabbit, human, pigeon, and hen. No significant differences among the five enzymes were detected in electrophoresis, ultracentrifugal analysis and kinetic properties. But some differences were observed in immunochemical investigation by means of double diffusion analysis and enzyme inhibition in the presence of antibodies against rat, human or pigeon muscle aldolase. Fowl anti-rat (or anti-human) muscle aldolase sera cross-reacted completely with other two mammalian aldolases, but did little with aldolases isolated from avians (hen and pigeon). Guinea pig anti-pigeon aldolase sera cross-reacted with hen aldolase, but did not with those of mammalians. It is concluded that in immunochemical and biochemical properties, three mammalian muscle aldolases are remarkably similar with each other, but those of birds lack some of the antigenic determinants present in the mammalian enzymes; even between hen and pigeon, the former aldolase lacks some of the determinants in that of the latter.