Muscle Aldolase Research Articles

ChemInform Abstract: α‐Amino Aldehyde Equivalents as Substrates for Rabbit Muscle Aldolase: Synthesis of 1,4‐Dideoxy‐1,4‐imino‐D‐arabinitol and 2(R),5(R) ‐Bis(hydroxymethyl)‐3(R),4(R)‐dihydroxypyrrolidine.

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The crystal structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster at 2.5A˚resolution

The structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster has been determined by X-ray diffraction at 2.5A˚resolution. The insect enzyme crystallizes in space group P2 12 12 1 with lattice constants a=86.60, b=116.80, c=151.58A˚. Molecular replacement with rabbit muscle aldolase as a search model has been employed to solve the structure. To improve the initial phases real space averaging, including phase extension from 4.0 to 2.5A˚, has been applied. Refinement of the atomic positions by molecular dynamics resulted in a crystallographic R-factor of 0.214. The tertiary structure resembles in most parts that of the vertebrate aldolase from rabbit muscle. Significant differences were found in surface loops and the N- and C-terminal regions of the protein. Here we present the first aldolase structure where the functionally important C-terminal arm is described completely.

Ketohexokinase (ATP: d-fructose 1-phosphotransferase) initiates fructose breakdown via the modified EMP pathway in halophilic archaebacteria

Abstract Ketohexokinase, catalysing the ATP-dependent phosphorylation of D -fructose to fructose 1-phosphate was identified as the enzyme responsible for the initiation of fructose breakdown via the modified EMP pathway in the halophilic arachaebacterium Haloarchla vallismortis . The phosphorylated product was identified as fructose 1-phosphate through its conversion to (i) a biphosphate ester by H. vallismortis 1-phosphofruktokinase, and (ii) trioses by rabbit muscle aldolase. The product of ketohexokinase reaction gave glyceraldehyde and dihydroxyacetone phosphate when cleaved directly by mammalian muscle aldolase, whereas, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate were produced when it was converted to the biphosphate ester prior to the treatment with aldolase. This is a first demonstration of ketohexokinase not only in an arachaebacterium but also in a prokaryote.

Long-range effects and conformational flexibility of aldolase

The conformational flexibility and long-range interactions in rabbit muscle aldolase induced by active-site ligand binding, cross-linking of the enzyme between Cys72 and Cys338, and removal of the C-terminal tyrosine residue were studied by following the changes in the microenvironments of Cys239 and Cys289 located outside the active site. It was found that substrates induced a conformational change in aldolase, which propagates from the active site to Cys239, which is located close to intersubunit contacts. The response of the enzyme is differential. Ligands having both C-1 and C-6 phosphates or C-1 phosphate only induce the enhancement of Cys239 reactivity, whereas those with C-6 phosphates only decrease Cys239 reactivity. This correlates well with a dramatic difference in kinetic parameters for a cleavage of fructose-1,6-P2 and fructose-1-P. Therefore, these changes can be interpreted as syncatalytic. Cross-linking of the aldolase subunit by an -S-S-bridge between Cys72 and Cys338 inactivates the enzyme, abolishes binding of active-site ligands, and induces a conformational change in the enzyme that can be detected far away (at Cys239 and Cys289) from the site of perturbation. Cys72 and Cys338 are not in the active site. This shows that the region of the active site and the environment of Cys72 and Cys338 are tightly coupled and that residues far away from the active site, through such coupling, can possess properties of active-site residues. Similar, although less dramatic changes are observed upon removal of the C-terminal tyrosine residue. In view of the results obtained in this paper, aldolase seems to be quite a flexible molecule, whose conformation is sensitive to the nature of a substrate bound to the enzyme and is able to transmit the information about a local perturbation over long distances within a molecule.

Spectroscopic evidence for NADH-induced conformational changes in rabbit muscle aldolase

The intrinsic fluorescence (steady-state spectra, anisotropy and nanosecond decay) in combination with phosphorescence at room temperature were used to detect and characterize conformational changes in rabbit muscle aldolase accompanying the NADH-binding process. Ligand binding has entailed a decrease in aldolase fluorescence intensity, a blue shift in its maximum and a polarization increase in a long wavelength part of the emission spectrum. The NADH binding induces the changes in room temperature phosphorescence - higher intensity and longer lifetime. The excited state energy transfer from tryptophans to NADH is not observed, and the character of spectroscopic changes on NADH binding allows us to reveal the spectroscopic heterogeneity among the tryptophan residues. The character, location of protein conformational changes associated with the binding of NADH and their relation to the tryptophans' microenvironment in aldolase are discussed.

.alpha.-Amino aldehyde equivalents as substrates for rabbit muscle aldolase: synthesis of 1,4-dideoxy-D-arabinitol and 2(R),5(R)-bis(hydroxymethyl)-3(R),4(R)-dihydroxypyrrolidine

.alpha.-Amino aldehyde equivalents as substrates for rabbit muscle aldolase: synthesis of 1,4-dideoxy-D-arabinitol and 2(R),5(R)-bis(hydroxymethyl)-3(R),4(R)-dihydroxypyrrolidine

Slow reversible inhibition of rabbit muscle aldolase by D-erythrulose 1-phosphate

Rabbit muscle aldolase was found to be inactivated in a slow, reversible manner by D-erythrulose 1-phosphate. This compound combined rapidly and reversibly with the enzyme to form an initial complex, which then only slowly (k i = 0.28 min −1) converted to a kinetically more stable form. This stable enzyme-ligand form was inactive toward the normal substrate of aldolase, fructose 1,6-bisphosphate. The inactive enzyme-ligand complex, however, could be decomposed (k r = 0.0041 min −1) to yield active enzyme once again by incubation in a solution devoid of D-erythrulose 1-phosphate.

Flexibility of the aldolase molecule measured using quenching-induced variations of the Forster distance for fluorescence energy transfer

The range of flexibility of the rabbit muscle aldolase molecule was studied using fluorescent labelled aldolase. The protein molecule was specifically labelled on the opposite sites of the enzyme subunit with the fluorescence energy donor and acceptor residues. Labelled aldolase with full enzymatic activity was used as a tool in the FRET studies between IAEDANS — donor or Cys-289 and IAF — acceptor on Cys-239. A range of Forster distance (R) was obtained by collisional quenching of the donor emission. The experiments of donor fluorescence quenching with wide range of acrylamide concentrations have shown the changes of donor-acceptor distances. In the absence of quencher the D-A distance distribution in characterized by an average value of 40.4 A, and a half-width of 0.13 A. A dramatic increase in half-width to 17.7A is observed after expositions of the enzyme to high acrylamide concentrations (0.13 M-0.68 M).

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 p k a values and/or reactivities than those of the soluble State.

ChemInform Abstract: Enzyme‐Catalyzed Reactions. Part 4. Aldolase‐Catalyzed C‐C Bond Formation for Stereoselective Synthesis of Nitrogen‐Containing Carbohydrates.

AbstractThe aldol addition reaction of the dihydroxyacetone monophosphate (I) with 3‐azido‐2‐hydroxypropanal (II) is catalyzed by rabbit muscle aldolase and yields stereoselectively the ketose 1‐phosphates isolated as the Ba‐salts (III) and (IV).

The interaction of rabbit muscle aldolase with NADH

Fluorescence studies on both the emission of aldolase and NADH bound to the enzyme were carried out. Aldolase was found to bind four molecules of NADH with K D = 6.0 ± 0.3 μ M . K D values for NADPH and NAD + were 41 ± 4 μ M and 140 ± 30 μ M , respectively. The affinity to NADH was comparable with that of some NAD-dependent dehydrogenases, and was not affected by the substrate or the inhibitor.

The crystal structure of human muscle aldolase at 3.0 Å resolution

The three-dimensional structure of fructose-1,6-bisphosphate aldolase from human muscle has been determined at 3.0 Å resolution by X-ray crystallography. The active protein is a tetramer of 4 identical subunits each of which is composed of an eight-stranded α/β-barrel structure. The lysine residue responsible for Schiff base formation with the substrate is located near the centre of the barrel in the middle of the sixth β-strand. While the overall topology of the α/β-barrel is very similar to those found in several other enzymes, the distribution of charged residues inside the core of the barrel seems distinct. The quaternary fold of human muscle aldolase uses interfacial regions also involved in the subunit association of other α/β-barrel proteins found in glycolysis, but exploits these regions in a manner not seen previously.

Aldolase-catalyzed C-C bond formation for stereoselective synthesis of nitrogen-containing carbohydrates

Rabbit muscle aldolase was found to catalyze stereoselective aldol addition of dihydroxyacetone phosphate (1) to 3-azido-2-hydroxypropana(2 ). The ketose 1-phosphates were isolated as barium salts, 4a/4b, and hydrolyzed with acid phosphatase. The mixture of 6-azido-6-deoxy-D-fructose(5) and 6-azido-6-deoxy-L-sorbose thus obtained was separated by anion-exchange chromatography. Reductive amination of 5 and 6 yielded, respectively, 1-deoxymannojirimycin (7) and 1-deoxynojirimycin (8), with high diastereoselectivity (>98:2). Analogous aldol addition of 1 to 3-azido-2-hydroxybutanal( 9)( E:Z = 92:8) afforded a mixture of the 6-azido-6,7-dideoxyheptuloses 12 and 13, which contained 88% of 6-azido-6,7-dideoxy-D-altro-heptulose(13 ). After anion-exchange chromatography, 13 was isolated as a 18:82 mixture of the β/α anomers. Reductive amination of pure 13 gave a mixture of 2,6,7-trideoxy-2,6-imino-D-glycero-D-manno and D-gluco-heptitol (14 and 15) (3:2 molar ratio), which likewise was separated by anion-exchange chromatography. If a mixture of 12 and 13 was hydrogenated under identical conditions, 2,6,7-trideoxy-2,6-imino-L-glycero-L-gulo-heptitol(16) could be isolated besides 14 and 15.

Stereospecific CC-bond formation with rabbit muscle aldolase - A chemoenzymatic synthesis of (+)-exo-brevicomin

(+)-(1 S,5 R,7 S)-Exo-brevicomin 9, a sex pheromone of the western pine bark beetle, is synthesized using an aldol reaction catalyzed by fructose-1,6-diphosphate aldolase (EC 4.1.2.13) from rabbit muscle as the key step by which the absolute configuration of the target is established.

The synthesis of aldose sugars from half-protected dialdehydes using rabbit muscle aldolase

The synthesis of aldose sugars from half-protected dialdehydes using rabbit muscle aldolase

Immobilization of pig muscle aldolase on a silica-based support

Pig muscle aldolase was covalently attached to a silica-based support possessing aldehyde functional groups. The activity of the immobilized enzyme was 37 U/g solid, and the specific activity calculated on a bound protein basis was 1.9 U/mg protein. The optimum pH for the catalytic activity was pH 7.5. The apparent optimum temperature was found to be 45 degrees C. The Km app value of the immobilized aldolase with D-fructose 1,6-diphosphate as substrate was 1.25 X 10(-4) M. The conformational stability was improved by the immobilization. The immobilized aldolase was used for the continuous splitting of D-fructose 1,6-diphosphate.

P-Nitrophenyl 3-diazopyruvate and diazopyruvamides, a new family of photoactivatable cross-linking bioprobes.

p-Nitrophenyl 3-diazopyruvate (DAPpNP) has been developed as a heterobifunctional cross-linking agent for synthesis of photoaffinity probes and photoactivatable cross-linking agents that are nucleophile specific. p-Nitrophenyl chloroglyoxylate is formed in high yield from oxalyl chloride and p-nitrophenol. Subsequent reaction with diazomethane produces DAPpNP in 50-60% overall yield. DAPpNP acylates primary and secondary amines to form 3-diazopyruvamides in high yields. 3-Diazopyruvamide derivatives have been formed from a wide variety of amines including aromatic amines, amino acids, and peptides. 3-Diazopyruvamides undergo photolysis and Wolff rearrangement at 300 nm to produce a ketene amide, which efficiently acylates nucleophilic species to form malonic acid amide derivatives. A family of photoactivatable 3-diazopyruvamide cross-linking agents was synthesized from amino acids. A cleavable, thiol-specific photoactivatable cross-linking agent was synthesized from cystamine. These reagents were caused to react with rabbit muscle aldolase to form mainly dimeric cross-linked species.

ChemInform Abstract: Rabbit Muscle Aldolase as a Catalyst in Organic Synthesis.

AbstractThe title catalyst RAMA is synthetically useful for the stereoselective aldol condensation between aldehydes having a wide range of structures and dihydroxyacetone phosphate (II).

Proteolysis of N-ethylmaleimide-modified aldolase loaded into erythrocyte ghosts: prevention by inhibitors of calpain

1. When rabbit muscle aldolase labelled with tritium and inactivated by N-ethylmaleimide (NEM) was loaded into erythrocyte ghosts, significant proteolysis of the loaded protein occurred. The major product of this proteolysis, separated by electrophoresis under dissociating conditions, was found to be approx. 2 kDa smaller than the parent protein. 2. Proteolysis was detectable during erythrocyte ghost loading at 0 degrees C, reaching a plateau after approx. 12 min. Subsequent incubation at 37 degrees C to allow resealing of the ghosts resulted in additional proteolysis, and up to 20% of the loaded protein was converted to the smaller 38 kDa derivative. 3. EDTA, EGTA, leupeptin and chymostatin, each inhibitors of calcium-activated neutral proteinases (calpains), were the most effective inhibitors of the proteolysis of NEM-inactivated aldolase in ghosts. Other proteinase inhibitors were ineffective, while phenylmethanesulphonyl fluoride was only partially effective. 4. Inhibition of the proteolysis by EGTA was prevented by CaCl2, supporting the involvement of erythrocyte calpain. 5. Pretreatment of ghosts with EGTA prior to loading of NEM-modified aldolase followed by microinjection of the protein into HeLa cells did not result in a different rate of its overall breakdown to acid-soluble products. EGTA is suggested as a useful agent for the erythrocyte ghost-mediated microinjection of calpain-sensitive proteins.

Enzymatic properties of fish muscle aldolase

1. Aldolases were isolated from the ordinary muscle of red sea bream Pagrus major, Pacific mackerel Scomber japonicus, and carp Cyprinus carpio by ammonium sulfate fractionation, followed by ion-exchange chromatography on DEAE-cellulose and CM-Sepharose CL-6B columns, and examined for enzymatic properties. 2. The aldolases showed the highest activity in a pH range from 6.8-7.8 Km values for fructose-1,6-bisphosphate ranged from 0.025-0.10 mM. 3. Irrespective of fish species, aldolase activity was inhibited by ATP, ADP, and AMP. ATP showed the strongest inhibition and was competitive with fructose-1,6-bisphosphate. 4. The aldolases did not require divalent metal ions for activation and were completely inhibited at 0.1 mM Cu2+. 5. Thermal inactivation of the enzymes was of the first-order reaction. Red sea bream, Pacific mackerel and carp enzymes lost the activity by 50% when incubated at 50 degrees C for 8, 14 and 23 min, respectively.