Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.     

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 × 10⁻⁶m and 2 × 10⁻³m 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 NaBH₄ 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.     

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.     

Subunit interface mutants of rabbit muscle aldolase form active dimers.

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.     

Predicted structure for aldolase.

Recent chromatographic and absorbance spectral measurements using the dye Cibacron blue F3GA (Stellwagen et al., 1975) have indicated that the substrate-binding site of fructose diphosphate aldolase is constructed by a supersecondary structural array closely resembling the NAD-domain commonly found in a variety of glycolytic enzymes. Analysis of the amino acid sequence of rabbit muscle aldolase according to the procedure of Chou &; Fasman (1974) predicts the occurrence of alternating β-strand and α-helical forming segments in the sequence region involving residues 147 to 299. Comparison of the sequence of residues 146 to 300 in aldolase with the sequence of residues 22 to 164 in dogfish lactate dehydrogenase which form its NAD-domain, suggests that the two sequence regions are related genetically. It is proposed that the locus of an NAD-domain in the structure of a protein can be predicted by sequence analysis provided that the protein specifically binds Cibacron blue F3GA.     

Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase.

The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.     

Properties of fructose 1,6-diphosphate aldolase from Drosophila melanogaster.

The amino acid composition and other properties of fructose 1,6-diphosphate aldolase from pupae of Drosophila melanogaster are reported and compared with those of other class I aldolases. Drosophila aldolase subunits contain only four residues of cysteine, five histidines, and two methionines. All four cysteine side chains react with 5,5′-dithiobis(2-nitrobenzoic acid) only in the presence of denaturating agent and are therefore thought to be buried within the molecule. With bromoacetate one carboxymethyl group is incorporated in the native enzyme with the loss of 90% of catalytic activity; inorganic phosphate is partially inhibiting this reaction. The near-uv absorption spectra of Drosophila and rabbit muscle aldolases are similar, the insect enzyme having higher absorbancies over the entire region corresponding to its higher tryptophan content. Circular dichroism-spectra of Drosophila aldolase indicate an α-helix content of 26%. Both the insect and vertebrate enzymes display marked tryptophan ellipticity bands between 290 and 300 nm.     

Fructose 1,6-diphosphate aldolase of Drosophila melanogaster: comparative sequence analyses of the cysteine-containing peptides.

Following tryptic digestion four cysteine-containing peptides per monomer have been isolated from fructose 1,6-diphosphate aldolase of Drosophila melanogaster. Sequence analyses of the peptides showed that three of the four cysteinyl residues appear to occur in homologous positions to three of the eight cysteines of rabbit muscle aldolase. Moreover they seem to be homologous also to three of the six sulfhydryl groups in sturgeon aldolase. The fourth cysteine-containing peptide of Drosophila aldolase has no homologous SH peptide either in the rabbit or in the sturgeon enzyme, but corresponds to another tryptic peptide in the rabbit aldolase. As deduced from homology all four SH peptides are localized in the buried region of the molecule. This conclusion is confirmed by the fact that all four cysteine-containing peptides have been isolated from the central cyanogen bromide fragment. Drosophila aldolase has no exposed thiol groups, thus demonstrating that these residues are not essential either in catalytic activity or for the stabilization of the three-dimensional structure.     

Analysis of the class I aldolase binding site architecture based on the crystal structure of 2-deoxyribose-5-phosphate aldolase at 0.99A resolution

The crystal structure of the bacterial (Escherichia coli) class I 2-deoxyribose-5-phosphate aldolase (DERA) has been determined by Se-Met multiple anomalous dispersion (MAD) methods at 0.99A resolution. This structure represents the highest-resolution X-ray structure of an aldolase determined to date and enables a true atomic view of the enzyme. The crystal structure shows the ubiquitous TIM alpha/beta barrel fold. The enzyme contains two lysine residues in the active site. Lys167 forms the Schiff base intermediate, whereas Lys201, which is in close vicinity to the reactive lysine residue, is responsible for the perturbed pK(a) of Lys167 and, hence, also a key residue in the reaction mechanism. DERA is the only known aldolase that is able to use aldehydes as both aldol donor and acceptor molecules in the aldol reaction and is, therefore, of particular interest as a biocatalyst in synthetic organic chemistry. The uncomplexed DERA structure enables a detailed comparison with the substrate complexes and highlights a conformational change in the phosphate-binding site. Knowledge of the enzyme active-site environment has been the basis for exploration of catalysis of non-natural substrates and of mutagenesis of the phosphate-binding site to expand substrate specificity. Detailed comparison with other class I aldolase enzymes and DERA enzymes from different organisms reveals a similar geometric arrangement of key residues and implies a potential role for water as a general base in the catalytic mechanism.     

The pyridoxal phosphate-binding site of rabbit muscle aldolase

Under appropriate conditions pyridoxal phosphate forms a Schiff-base derivative with a specific lysine residue in rabbit muscle aldolase, with the incorporation of slightly less than 1 equiv of pyridoxal phosphate per enzyme subunit. Reduction of the Schiff base with tritium-labeled borohydride introduces a radioactive label at this site. A tryptic peptide containing the labeled lysine residue has been isolated and found to possess the following sequence: Gly-Gly-Val-Val-Gly-Ile-Lys¹-Val-Asp-Lys, where the asterisk indicates the modified lysine residue.     

Hydroxynaphthaldehyde phosphate derivatives as potent covalent Schiff base inhibitors of fructose-1,6-bisphosphate aldolase

Interactions of phosphate derivatives of 2,6-dihydroxynaphthalene (NA-P(2)) and 1,6-dihydroxy-2-naphthaldehyde (HNA-P, phosphate at position 6) with fructose-1,6-bisphosphate aldolase from rabbit muscle were analyzed by enzyme kinetics, difference spectroscopy, site-directed mutagenesis, mass spectrometry, and molecular dynamics. Enzyme activity was competitively inhibited by NA-P(2), whereas HNA-P exhibited slow-binding inhibition with an overall inhibition constant of approximately 24 nM. HNA-P inactivation was very slowly reversed with t(1/2) approximately 10 days. Mass spectrometry and spectrophotometric absorption indicated that HNA-P inactivation occurs by Schiff base formation. Rates of enzyme inactivation and Schiff base formation by HNA-P were identical and corresponded to approximately 4 HNA-P molecules bound par aldolase tetramer at maximal inhibition. Site-directed mutagenesis of conserved active site lysine residues 107, 146, and 229 and Asp-33 indicated that Schiff base formation by HNA-P involved Lys-107 and was promoted by Lys-146. Titration of Lys-107 by pyridoxal 5-phosphate yielded a microscopic pK(a) approximately 8 for Lys-107, corroborating a role as nucleophile at pH 7.6. Site-directed mutagenesis of Ser-271, an active site residue that binds the C(1)-phosphate of dihydroxyacetone phosphate, diminished HNA-P binding and enabled modeling of HNA-P in the active site. Molecular dynamics showed persistent HNA-P phosphate interactions with the C(1)-phosphate binding site in the noncovalent adduct. The naphthaldehyde hydroxyl, ortho to the HNA-P aldehyde, was essential for promoting carbinolamine precursor formation by intramolecular catalysis. The simulations indicate a slow rate of enzyme inactivation due to competitive inhibition by the phenate form of HNA-P, infrequent nucleophilic attack in the phenol form, and significant conformational barrier to bond formation as well as electrostatic destabilization of protonated ketimine intermediates. Solvent accessibility by Lys-107 Nz was reduced in the covalent Schiff base complex, and in those instances where water molecules interacted with Lys-107 in the simulations, Schiff base hydrolysis was not mechanistically favorable. The findings at the molecular level corroborate the observed mechanism of slow-binding tight inhibition by HNA-P of muscle aldolase and should serve as a blueprint for future aldolase inhibitor design.     

Radiation inactivation of rabbit muscle aldolase.

Pulse radiolysis and steady-state X-radiolysis have been used to investigate the radiation inactivation of aldolase from rabbit muscle. Both eaq-and OH readily react with aldolase, and contribute to inactivation. The radical anions (CNS)2-and (Br)2-react with aldolase at neutral pH. The progressive addition of alkali results in an increase in the second-order rate constants, with an apparent pK approximately 10 +/- 0-3, and with the formation of an unstable intermediate, lambdamax approximately 400 nm resembling a phenoxyl radical. Steady-state radiolysis in the presence of (CNS)2-and (Br)2- at alkaline pH results in increased aldolase inactivation, with a pK of enzyme inactivation similar to that observed for reaction of the radical anions. We propose that a reaction of the radical anoins with tyrosine residues accounts for the resultant inactivation.     

Equilibrium partition studies of the interaction between aldolase and myofibrils

The adsorption of aldolase to myofibrils derived from rabbit skeletal muscle has been investigated by partition equilibrium studies at pH 6.8, I = 0.158 M, and the results interpreted in terms of an intrinsic association constant of 410,000 m^?1 for the interaction of four sites on aldolase with myofibrillar sites, there being one such site for every 10–12 heptameric repeat units of F-actin-tropomyosin-troponin thin filament. Involvement of the active site of the enzyme in the adsorption process is indicated by the fact that competitive inhibition of the phenomenon by phosphate may be accounted for by an intrinsic association constant of 400 m^?1 for the aldolase-phosphate interaction, a value in good agreement with that describing phosphate inhibition of the enzymatic hydrolysis of fructose-1,6-bisphosphate under similar conditions. On the basis of these equilibrium constants plus the aldolase and thin filament contents of muscle, resting muscle is indicated as containing a significant proportion (25–30%) of aldolase in the bound form, with changes in the subcellular distribution of the enzyme being likely during exercise due to the increased concentrations of Ca²⁺ and fructose-1,6-bisphosphate that then prevail.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Site-specific modification or rabbit muscle aldolase with fluorescent probes

The site-specific modification of rabbit muscle aldolase A by labeling of thiol residues of Cys-289 with 5-(2-((iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid and Cys-239 with 5-iodoacetamidofluorescein or 4-dimethylamino-phenylazophenyl-4'-maleimide has been described. The method is based on the differences in kinetics of the chemical modification of aldolase thiols with the above reagents either in the presence or in the absence of a competitive inhibitor. The spectral properties of the doubly labeled aldolase derivatives were compared with those of the singly labeled enzyme. The doubly labeled aldolase derivatives exhibited full catalytic activity.     

Photoaffinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 8-azido-1,N6-ethenoadenosine 5'-triphosphate

Steady-state kinetic measurements have shown that 8-azido-1,N6-ethenoadenosine 5'-triphosphate (8-N3-epsilon ATP) can be noncovalently bound to rabbit muscle fructose 1,6-bisphosphate aldolase with Ki = 0.075 mM at pH 8.5. This binding is purely competitive with substrate and occurs at the strong binding site for mononucleotides. Photoaffinity labeling of aldolase in the presence of 8-azido-1,N6-ethenoadenosine 5'-triphosphate results in inactivation of the enzyme. Aldolase is protected against modification in the presence of the inhibitors hexitol 1,6-bisphosphate or ATP. The labeling is saturable, and a good correlation is observed between the loss of enzymatic activity and the incorporation of 8-N3-epsilon ATP into aldolase. In addition, aldolase loses its ability to bind to phosphocellulose following modification. Digestion of labeled protein with trypsin, chymotrypsin, and cyanogen bromide revealed substantial modification of peptide 259-269. Thr-265 was identified as the residue that was covalently modified by 8-N3-epsilon ATP. On the basis of these results and other data we propose a model for the mononucleotide binding site.     

Purification and properties of rabbit heart muscle aldolase.

Fructose diphosphate aldolase (D-fructose-1,6-biphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit heart has been purified and obtained in crystalline form. The preparations are homogeneous on the basis of disc gel electrophoresis and ultracentrifugation. The catalytic and the molecular properties indicate that this is aldolase A. A comparison was made between rabbit heart aldolase and the rabbit muscle enzyme. The sedimentation coefficient, energy of activation and Michaelis constant for Fru-1,6-P2 were found to be identical with the values obtained for the muscle enzyme. As in case of the muscle enzyme, heart aldolase was found to have a broad pH optimum, remarkable stability over a wide pH range, and the ability to form a Schiff base intermediate with dihydroxyacetone phosphate upon reduction with borohydride. Cleavage of the methionyl bonds with CNBr yields the same pattern as obtained with the muscle enzyme.     

A hydrophobic pocket in the active site of glycolytic aldolase mediates interactions with Wiskott-Aldrich syndrome protein

Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, four-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds around the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C terminus with its active site and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure, a novel naphthol phosphate-based inhibitor of aldolase was identified, and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrich syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.