Studies on the structure of rabbit muscle aldolase. 3. Primary structure of the BrCN peptide containing the active site

The four peptide segments obtained from rabbit muscle aldolase by cleavage with BrCN and separation with gel-filtration chromatography (1) have been redesignated according to their positions in the molecule, N-A-B-C. The primary structure of segment A, containing 66 amino acid residues, including the Schiff base-forming lysine at the active site, has been elucidated by isolation and sequence analyses of the proteolytic subfragments. Preliminary separation of tryptic peptides containing 7–25 residues was achieved by chromatography on Sephadex G-25 which facilitated subsequent purification. For the study of the tryptic peptide of 25 residues further fragmentation with pepsin then subtilisin (Nagarse) was employed. Edman degradation directly after subtilisin cleavage of a peptide was found useful in avoiding deamidation of a glutamine NH₂-terminus newly formed in the proteolysis. The sequence of 90 amino acids in the center region of the polypeptide chain of rabbit muscle aldolase has now been established.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Studies on the structure of rabbit muscle aldolase: Ordering of the tryptic peptides; Sequence of 164 amino acid residues in the NH2-terminal BrCN peptide

The sequence of 164 amino acid residues in the NH₂-terminal BrCN peptide of rabbit muscle aldolase has been determined. The information has permitted location of the following amino acid residues involved in the catalytic activity or in maintaining the structural integrity of the enzyme: Cys-72, forms a disulfide bridge with Cys-336 in the COOH-terminal segment on inactivation of the enzyme by oxidation; Lys-107, forms a Schiff base with pyridoxal phosphate upon inactivation of aldolase by this reagent; Cys-134 and Cys-177, buried, do not react with SH-reagents in the native enzyme.     

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.     

The amino acid sequence of the C- terminal cyanogen bromide peptide of human J chain

The carboxyl-terminal peptide obtained from human J chain treated with cyanogen bromide (CNBr)¹ has been isolated and the amino acid sequence determined as Val - Glx - Thr - Ala - Leu - Thr - Pro - Asx - Ala - CMCys - Tyr - Pro - Asx. Comparisons with the primary structures of human lambda, kappa, alpha, or mu chains failed to disclose analogous regions with these immunoglobulins.     

Primary structure at the active sites of beef and rabbit liver aldolases

The sequence of the tryptic heptaeicosapeptide isolated from the active site of beef liver aldolase has been determined and shown to be identical with that of the corresponding peptide from rabbit liver aldolase. The sequence is Ala-Leu-Asn-Asp-His-His-Val-Tyr-Leu-Glu-Gly-Thr-Leu-Leu-βGlys-Pro-Asn-Met-Val-Thr-Ala-Gly-His-Ala-Cys-Thr-Lys. There is extensive homology with peptides isolated from the same region of a variety of muscle aldolases.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

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.     

Comparative study of the phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophlus by the tritium topography method

A comparative study of thermostability and aminoacid composition of the phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. Compared with the mesophilic enzyme, a considerable increase of Pro, Leu, Phe, Arg and decrease of Asx, Ile, Ser, Thr and Lys content have been revealed in the thermophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of both the enzymes. In the E. coli enzyme, Thr residues were also easy to access while on the surface of the thermophilic enzyme there were more Arg residues. The quantitative assay of the surface compositions revealed the increased exposure of the (Leu + Ile) residues on the thermophilic protein as well as of the charged Asx and Arg residues. A possible correlation of the observed effects with thermostability is discussed.     

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.     

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.     

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.     

Comparison of repetitive sequences derived from high molecular weight subunits of wheat glutenin, an elastomeric plant protein

A strategy has been developed to create repetitive peptides incorporating substitutions in the PGQGQQGYYPTSLQQ consensus repeat sequence of high molecular weight subunits in order to investigate natural sequence variations in elastomeric proteins of wheat gluten. After introduction of glutamic and aspartic acid residues, the peptide behaved similarly to the unmodified form at low pH, but became readily water soluble at pH > 6. Substitution of Gln for Leu at position 13 resulted in only small changes to the secondary structure of the water-insoluble peptides, as did Tyr8His and Thr11Ala. The effects of proline substitutions depended on their location: Leu13Pro substitution had little effect on solubility and structure, but Gln6Pro substitution resulted in dramatic changes. Peptides with two Gln6Pro substitutions had similar properties to the water-insoluble parental peptide, but those with 6 or 10 substitutions were readily soluble. The results indicated that specific sequences influence noncovalent intermolecular interactions in wheat gluten proteins.     

Human transferrins C and DChi: An amino acid difference

A single peptide difference has been found in tryptic digests of human transferrins C and D_Chi. The peptide isolated from Tf C was shown to have the sequence Asp-Ser-Ala-His-Gly-Phe-Leu-Lys. The corresponding peptide from Tf D_Chi has the composition (Gly, Phe, Leu, Lys). Apparently, histidine of the Tf C peptide has been replaced by lysine or arginine in Tf D_Chi, producing a new point of attack for trypsin. On the basis of the genetic code, arginine is proposed as the replacement.Supported by U.S. Public Health Service grants # GM 09326, # 5-K3-GM-18,381, and # GM 00337 from the National Institutes of Health. From the Department of Zoology, The University of Texas, Austin.     

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.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Human skeletal-muscle aldolase: N-terminal sequence analysis of CNBr- and o-iodosobenzoic acid-cleavage fragments

Fructose-1,6-bisphosphate aldolase was purified from human skeletal-muscle by affinity elution chromatography. Four CNBr-cleavage fragments were purified by gel filtration, and their N-terminal amino acid sequences were determined. Cleavage with o-iodosobenzoic acid at the three tryptophan residues also yielded fragments suitable for N-terminal sequence analysis. Thus, the sequence of 272 of the 363 residues was established. These sequence results allow many of the discrepancies between the two published rabbit skeletal-muscle aldolase sequences to be resolved. The human aldolase sequence reported here is 96% identical to a "consensus" rabbit aldolase sequence. A comparison with a partial sequence of Drosophila aldolase (103 residues) shows 80% identity. The determination of the amino acid sequence of human aldolase is important for the interpretation of the crystal structure of this enzyme.     

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.     

Amino acid sequence of rabbit skeletal muscle myosin light chain kinase

The amino acid sequence of the amino-terminal, 235-residue segment of rabbit skeletal muscle myosin light chain kinase has been determined. Together with the carboxyl-terminal segment previously described [Takio, K., Blumenthal, D. K., Edelman, A. M., Walsh, K. A., Krebs, E. G., & Titani, K. (1985) Biochemistry 24, 6028], the present work completes the 603-residue sequence of this protein. The amino-terminal segment that has been analyzed herein corresponds to a domain reported to be of highly asymmetrical shape and as yet unknown function. Secondary structure calculations failed to provide any evidence of alpha-helix or beta-structures, but polyproline II like helical structure is possible. Sequence analysis indicates the presence of approximately equal quantities of two isoforms differing in a single amino acid replacement. Unexpected difficulties were encountered in the present sequence analysis due to the presence of acid-labile Asp-Pro bonds and to five separable variants of a blocked 21-residue amino-terminal peptide, arising from rearrangement at an Asn-Gly bond.     

The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones

The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81% amino acid homology with aldolase A and 70% homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons separated by eight introns, all in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.