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.     

Human aldolase isozyme gene: the structure of multispecies aldolase B mRNAs.

A complete nucleotide sequence of human aldolase B mRNA was determined with a recombinant cDNA (pHABL120-3). The cDNA insert was composed of 1,652 bases excluding poly(A) tail and the sequence was consistent with the previous results reported by others. However, S1 nuclease mapping and subsequent genomic analysis allowed us to know that the clone possesses two more sites corresponding to 5'-termini in the 5'-noncoding region and another site of polyadenylation in the 3'-noncoding region. In fact, the major aldolase B mRNA species occupying 90% of the total mRNAs initiated at the predominant position corresponding to the position around -82 of the 5'-noncoding sequence in pHABL120-3 and terminated at the distal polyadenylation site. Second species accounting for 9% of the mRNAs initiated at the same site and terminated at the proximal polyadenylation site. The remainings have a longer 5'-noncoding sequence which starts from further upstream region of the major one and pHABL120-3 corresponds to one of these largest clones.     

Effect of acrylamide on aldolase structure. II. Characterization of aldolase unfolding intermediates.

Molecules of muscle aldolase A exposed to acrylamide change their conformation via I1, T, I2, D intermediates [1] and undergo a slow irreversible chemical modification of thiol groups. There is no direct correlation between activity loss and thiol groups modification. In the native enzyme two classes of Trp residues of 1. 8 ns and 4.9 ns fluorescence lifetime have been found. Acrylamide (0. 2-0.5 M) increases lifetime of longer-lived component, yet the transfer of aldolase molecules even from higher (1.0 M) perturbant concentration to a buffer, allows regain original Trp fluorescence lifetime. I1, detected at about 0.2 M acrylamide, represents low populated tetramers of preserved enzyme activity. T, of maximum population at about 0.7-1.0 M acrylamide, consists of meta-stable tetramers of partial enzymatic activity. These molecules are able to exchange their subunits with aldolase C in opposition to the native molecules. At transition point for I2 appearance (1.8 M acrylamide), aldolase becomes highly unstable: part of molecules dissociate into subunits which in the absence of perturbant are able to reassociate into active tetramers, the remaining part undergoes irreversible denaturation and aggregation. Some expansion of aldolase tetramers takes place prior to dissociation. D, observed above 3.0 M acrylamide, consists of irreversibly denatured enzyme molecules.     

Subunit structure of rabbit brain aldolase

Rabbit brain contains a mixture of aldolase A (muscle type) and aldolase C (brain type), present largely as the hybrid forms A₃C, A₂C₂, and AC₃, with smaller amounts of the homopolymers A₄ and C₄. We have developed new procedures for the isolation of the A-C hybrid set and the aldolase C subunits and compared the structure of these subunits with those of aldolase A. The two isoenzymes differ significantly in amino acid composition, but each contains three methionine residues per subunit and yields four peptides on cleavage with cyanogen bromide. The three methionine residues appear to occupy similar positions in the polypeptide chains but the molecular weight of the aldolase C subunit is only 37,000, approximately 10% smaller than that of the subunit of aldolase A. The difference is attributable to two or more deletions, totaling 30–40 amino acid residues, in two of the four BrCN peptides. The deletions include two of the buried cysteine residues that are located in the center of the polypeptide chain in aldolase A; these residues in aldolase A are, therefore, not involved in the contacts between the subunits in the tetramer. Aldolase C also lacks several of the histidine residues that are located near the active-site lysine residue of aldolase A, thus excluding these residues from participation in the catalytic mechanism.     

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.     

Effect of acrylamide on aldolase structure. I. Induction of intermediate states.

Acrylamide is a fluorescence quencher frequently applied for analysis of protein fluorophores exposure with the silent assumption that it does not affect the native structure of protein. In this report, it is shown that quenching of tryptophan residues in aldolase is a time-dependent process. The Stern-Volmer constant increases from 1.32 to 2.01 M-1 during the first 100 s of incubation of aldolase with acrylamide. Two tryptophan residues/subunit are accessible to quenching after 100 s of aldolase interaction with acrylamide. Up to about 1.2 M acrylamide concentration enzyme inactivation is reversible. Independent analyses of the changes of enzyme activity, 1ANS fluorescence during its displacement from aldolase active-site, UV-difference spectra and near-UV CD spectra were carried out to monitor the transition of aldolase structure. From these measurements a stepwise transformation of aldolase molecules from native state (N) through intermediates: I1, T, I2, to denatured (D) state is concluded. The maxima of I1, T, I2 and D states populations occur at 0.2, 1.0, 2.0 and above 3.0 M of acrylamide concentration, respectively. Above 3.5 M, acrylamide aldolase molecules become irreversibly inactivated.     

The low-resolution structure of human muscle aldolase

The three-dimensional structure of human muscle aldolase has been solved at 5 A resolution with the use of two isomorphous heavy atom derivatives. The enzyme's four subunits are arranged about three mutually perpendicular intersecting twofold axes to form a compact spherical molecule. The subunit boundaries are clearly defined but a possible domain structure is not apparent in this preliminary electron density map.     

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.     

The reactivity of thiol groups and the subunit structure of aldolase

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-(14)C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5'-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.     

Characterization and crystal structure of Escherichia coli KDPGal aldolase

2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of k(cat)/K(M) and k(cat) of 1.9x10(4)M(-1)s(-1) and 4s(-1), respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4A and displays the same (alpha/beta)(8) topology, as KDPG aldolase. We have also determined a 2.1A structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.