Amino-acid Sequence Homology in the Muscle Aldolases from Sturgeons of Different Species

STUDIES on the primary structure of aldolases isolated from ox, pig and rabbit muscle show that the amino-acid sequence of fructose 1,6-diphosphate aldolase [EC 4.1.2.13] has been highly conserved throughout mammalian evolution¹. But comparison of the primary structure of the enzyme from these species with that from the muscle of a single North Sea sturgeon, presumably Acipenser sturio, indicated that although the proteins were homologous, a number of amino-acid replacements occurred between sturgeon aldolase and the aldolases of the phylogenetically distant mammalian species¹. As a knowledge of the nature and number of amino-acid replacements between homologous proteins caft provide information both about the functional role of individual residues and about evolution, further comparative studies of rabbit and sturgeon aldolases were undertaken and an account of the sequence homology around the active-site-lysine residue of aldolases from rabbit muscle, rabbit liver and the muscle of the river sturgeon of Eastern Canada, Acipenser fulvescens, has been given^2,3.     

Fructose diphosphate aldolase from Mycobacterium smegmatis. Functional similarities with rabbit muscle aldolase.

Fructose diphosphate aldolase of Mycobacterium smegmatis is found to be a class I type aldolase and possesses functional similarities with rabbit muscle aldolase with respect to the amino acid residues at the catalytic site. The presence of a lysine residue at the active site is indicated by the formation of a Schiff-base with the substrate. The lower degree of inactivation compared to rabbit muscle aldolase on treatment with carboxypeptidase-A suggests the absence of an essential terminal tyrosine residue. Participation of histidine residues in enzyme catalysis is suggested by the photoinactivation of the enzyme in presence of methylene blue. Finally, thiol groups do not seem to have a direct role in catalysis.     

Limited proteolysis of liver and muscle aldolases: Effects of subtilisin,cathepsin B,and Staphylococcus aureus protease

Limited proteolysis of rabbit liver and muscle aldolases by subtilisin and cathepsin B results in decreased catalytic activity, associated with the release of acid-soluble peptides from the COOH terminus. Analysis of the sequence of these peptides confirms the COOH-terminal sequence of the muscle enzyme and provides new information on the COOH-terminal sequence of the liver enzyme. As previously reported for muscle aldolase, cathepsin B releases mainly dipeptides from the COOH terminus of liver aldolase. The COOH-terminal sequence of rabbit liver aldolase is SerThrGlnSerLeuPheThrAla SerTyrThrTyr. The Gln-Ser bond is resistant to Staphylococcus aureus protease, which hydrolyzes a GluSer bond at the corresponding positions in the muscle enzyme.     

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.     

Location and primary structure of the substrate binding site peptide of aldolase C

An octadecapeptide containing the substrate-combining site of rabbit brain aldolase (aldolase C) has been isolated. This peptide has tentatively been assigned the structure: Ala-Leu-Ser(Asx,His,His,Val,Tyr)(Leu,Glx,Gly,Thr,Leu,Leu)(Lys,Pro,Asx,Met). The primary sequence of this peptide thus appears to be very similar to that of the active-site peptide of rabbit muscle aldolase (aldolase A), but it is located in a different BrCN segment, approximately 50 residues closer to the NH₂-terminus of the aldolase C subunit. A tentative sequence has also been obtained for an adjacent nonapeptide, also homologous with the corresponding structure in aldolase A. The evidence suggests that a large segment of the peptide chain in aldolase C may be translocated, as compared with aldolase A.     

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.     

Investigation of the role of lysine in the subunit contact regions of rabbit muscle aldolase.

Rabbit muscle aldolase (E.C. 4. 1. 2. 13) was guanidinated by reaction with O-methylisourea. Up to 60% of the lysine residues can be guanidinated without any dissociation of the tetramer but with a complete loss of enzymatic activity. Native and guanidinated aldolase can be dissociated into monomers in 2.4 m MgCl₂ with only slight change in conformation of the subunit. Nitrotroponylation of guanidinated aldolase in dilute buffer gives no reaction whereas in 2.4 m MgCl₂ nitrotroponlylation modifies another 8–12% of the lysine residues. Removal of MgCl₂ by dialysis affords 100% recovery of activity and tetrameric structure for native aldolase and 100% recovery of tetrameric structure for guanidinated aldolase. In contrast nitrotroponylated and guanidinated aldolase remains monomeric before precipitating as the MgCl₂ concentration is lowered. It is concluded that lysine may be involved in the protein-protein interaction of the subunit contact domains of muscle aldolase.     

The NAD domain and the predicted structure for aldolase: a word of caution.

The proposal of E. Stellwagen [(1976) J. Mol Biol., 106, 903–911] that the structure of a protein can be predicted by sequence analysis provided that the protein specifically binds Cibacron blue F3GA, is not sound at least for muscle fructose bisphosphate aldolase. Contrary to the predictions we have shown that Cibacron blue does not interact directly with lysine 227 at the catalytic sites but with different sites which bind also ATP and fructose bisphosphate. We have shown also that aldolase binds 3.5 molecules of dye per subunit (dissociation constant 1.9 μm), too great a number to support the hypothesis that the binding of Cibacron blue is a specific indication of the presence of an NAD domain.     

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.     

Reactivity of the active-centre lysine residue of rabbit muscle aldolase

The method of competitive labelling with [(3)H]acetic anhydride as the labelling reagent was used to determine the properties of the active-centre lysine residue of rabbit muscle aldolase. This residue is much less reactive than a normal exposed lysine residue towards this reagent, and its reactive properties did not parallel the pH-activity profile for aldolase. At higher pH values it became reactive, but this was shown to be due to disruption of the enzyme structure. The binding of the competitive inhibitor phosphate did not alter the reactive properties. It is concluded that the active-centre lysine has an apparent pK(a) greater than 11.5 and probably is made nucleophilic during the catalytic process, perhaps by proton abstraction.     

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.     

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.     

Evolutionary conserved N-terminal region of human muscle fructose 1,6-bisphosphatase regulates its activity and the interaction with aldolase

N-terminal residues of muscle fructose 1,6-bisphosphatase (FBPase) are highly conserved among vertebrates. In this article, we present evidence that the conservation is responsible for the unique properties of the muscle FBPase isozyme: high sensitivity to AMP and Ca(2+) inhibition and the high affinity to muscle aldolase, which is a factor desensitizing muscle FBPase toward AMP and Ca(2+). The first N-terminal residue affecting the affinity of muscle FBPase to aldolase is arginine 3. On the other hand, the first residue significantly influencing the kinetics of muscle FBPase is proline 5. Truncation from 5-7 N-terminal residues of the enzyme not only decreases its affinity to aldolase but also reduces its k-(cat) and activation by Mg(2+), and desensitizes FBPase to inhibition by AMP and calcium ions. Deletion of the first 10 amino acids of muscle FBPase abolishes cooperativity of Mg(2+) activation and results in biphasic inhibition of the enzyme by AMP. Moreover, this truncation lowers affinity of muscle FBPase to aldolase about 14 times, making it resemble the liver isozyme. We suggest that the existence of highly AMP-sensitive muscle-like FBPase, activity of which is regulated by metabolite-dependent interaction with aldolase enables the precise regulation of muscle energy expenditures and might contributed to the evolutionary success of vertebrates.     

The crystal structure of human muscle aldolase at 3.0 A resolution

The three-dimensional structure of fructose-1,6-bisphosphate aldolase from human muscle has been determined at 3.0 A resolution by X-ray crystallography. The active protein is a tetramer of 4 identical subunits each of which is composed of an eight-stranded alpha/beta-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 beta-strand. While the overall topology of the alpha/beta-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 alpha/beta-barrel proteins found in glycolysis, but exploits these regions in a manner not seen previously.     

Structural Similarities between Spinach Chloroplast and Cytosolic Class I Fructose 1,6-Bisphosphate Aldolases : Immunochemical and Amino-Terminal Amino Acid Sequence Analysis

Immunochemical studies using polyclonal antisera prepared individually against highly purified cytosolic and chloroplast spinach leaf (Spinacia oleracea) fructose bisphosphate aldolases showed significant cross reaction between both forms of spinach aldolase and their heterologous antisera. The individual cross reactions were estimated to be approximately 50% in both cases under conditions of antibody saturation using a highly sensitive enzyme-linked immunosorbent assay. In contrast, the class I procaryotic aldolase from Mycobacterium smegmatis and the class II aldolase from yeast (Saccharomyces cerevisiae) did not cross-react with either type of antiserum. The 29 residue long amino-terminal amino acid sequences of the procaryotic M. smegmatis and the spinach chloroplast aldolases were determined. Comparisons of these sequences with those of other aldolases showed that the amino-terminal primary structure of the chloroplast aldolase is much more similar to the amino-terminal structures of class I cytosolic eucaryotic aldolases than it is to the corresponding region of the M. smegmatis enzyme, especially in that region which forms the first “beta sheet” in the secondary structure of the eucaryotic aldolases. Moreover, results of a systematic comparison of the amino acid compositions of a number of diverse eucaryotic and procaryotic fructose bisphosphate aldolases further suggest that the chloroplast aldolase belongs to the eucaryotic rather than the procaryotic “family” of class I aldolases.     

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.     

Active-site labelling of triose phosphate isomerase. The reaction of bromohydroxyacetone phosphate with a unique glutamic acid residue and the migration of the label to tyrosine

Triose phosphate isomerase from chicken muscle reacts stoicheiometrically with the active-site-directed irreversible inhibitor bromohydroxyacetone phosphate with concomitant loss of all catalytic activity. The primary site of attachment has been shown to be a unique glutamic acid residue in the sequence Ala-Tyr-Glu-Pro-Val-Trp. Unless the inhibitor-enzyme bond is stabilized by reduction of the C-2 carbonyl group with borohydride, the phosphate group is lost and the label migrates to the adjacent tyrosine residue. It is suggested that the gamma-carboxylate group of the glutamic acid residue may be the base responsible for primary proton abstraction from substrate in the catalysis. The failure of this reagent specifically to inactivate either muscle or yeast aldolase, and the use of the reagent in preparing isomerase-free glycolytic enzymes, is discussed.     

Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver.

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.