Fructose metabolism as a common evolutionary pathway of survival associated with climate change, food shortage and droughts.

Mass extinctions occur frequently in natural history. While studies of animals that became extinct can be informative, it is the survivors that provide clues for mechanisms of adaptation when conditions are adverse. Here, we describe a survival pathway used by many species as a means for providing adequate fuel and water, while also providing protection from a decrease in oxygen availability. Fructose, whether supplied in the diet (primarily fruits and honey), or endogenously (via activation of the polyol pathway), preferentially shifts the organism towards the storing of fuel (fat, glycogen) that can be used to provide energy and water at a later date. Fructose causes sodium retention and raises blood pressure and likely helped survival in the setting of dehydration or salt deprivation. By shifting energy production from the mitochondria to glycolysis, fructose reduced oxygen demands to aid survival in situations where oxygen availability is low. The actions of fructose are driven in part by vasopressin and the generation of uric acid. Twice in history, mutations occurred during periods of mass extinction that enhanced the activity of fructose to generate fat, with the first being a mutation in vitamin C metabolism during the Cretaceous-Paleogene extinction (65 million years ago) and the second being a mutation in uricase that occurred during the Middle Miocene disruption (12-14 million years ago). Today, the excessive intake of fructose due to the availability of refined sugar and high-fructose corn syrup is driving 'burden of life style' diseases, including obesity, diabetes and high blood pressure.

Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate.

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.

The structure of human liver fructose-1,6-bisphosphate aldolase.

The X-ray crystallographic structure of the human liver isozyme of fructose-1,6-bisphosphate aldolase has been determined by molecular replacement using a tetramer of the human muscle isozyme as a search model. The liver aldolase (B isozyme) crystallized in space group C2, with unit-cell parameters a = 291.1, b = 489.8, c = 103.4 A, alpha = 90, beta = 103.6, gamma = 90 degrees. These large unit-cell parameters result from the presence of 18 subunits in the asymmetric unit: four catalytic tetramers and a dimer from a fifth tetramer positioned on the twofold crystallographic axis. This structure provides further insight into the factors affecting isozyme specificity. It reveals small differences in secondary structure that occur in regions previously determined to be isozyme specific. Two of these regions are at the solvent-exposed enzyme surface away from the active site of the enzyme. The most significant changes are in the flexible C-terminal region of the enzyme, where there is an insertion of an extra alpha-helix. Point mutations of the human liver aldolase are responsible for the disease hereditary fructose intolerance. Sequence information is projected onto the new crystal structure in order to indicate how these mutations bring about reduced enzyme activity and affect structural stability.

Structure of a fructose-1,6-bis(phosphate) aldolase liganded to its natural substrate in a cleavage-defective mutant at 2.3 A(,).

Class I fructose-1,6-bis(phosphate) aldolase is a glycolytic enzyme that catalyzes the cleavage of fructose 1,6-bis(phosphate) through a covalent Schiff base intermediate. Although the atomic structure of this enzyme is known, assigning catalytic roles to the various enzymic active-site residues has been hampered by the lack of a structure for the enzyme-substrate complex. A mutant aldolase, K146A, is unable to cleave the C3-C4 bond of the hexose while retaining the ability to form the covalent intermediate, although at a greatly diminished rate. The structure of rabbit muscle K146A-aldolase A, in complex with its native substrate, fructose 1,6-bis(phosphate), is determined to 2.3 A resolution by molecular replacement. The density at the hexose binding site differs between subunits of the tetramer, in that two sites show greater occupancy relative to the other two. The hexose is bound in its linear, open conformation, but not covalently linked to the Schiff base-forming Lys-229. Therefore, this structure most likely represents the bound complex of hexose just after hemiketal hydrolysis and prior to Schiff base formation. The C1-phosphate binding site involves the three backbone nitrogens of Ser-271, Gly-272, and Gly-302, and the epsilon-amino group of Lys-229. This is the same binding site previously found for the analogous phosphate of the product DHAP. The C6-phosphate binding site involves three basic side chains, Arg-303, Arg-42, and Lys-41. The residues closest to Lys-229 were relatively unchanged in position when compared to the unbound wild-type structure. The major differences between the bound and unbound enzyme structures were observed in the positions of Lys-107, Arg-303, and Arg-42, with the greatest difference in the change in conformation of Arg-303. Site-directed mutagenesis was performed on those residues with different conformations in bound versus unbound enzyme. The kinetic constants of these mutant enzymes with the substrates fructose 1, 6-bis(phosphate) and fructose 1-phosphate are consistent with their ligand interactions as revealed by the structure reported here, including differing effects on k(cat) and K(m) between the two substrates depending on whether the mutations affect C6-phosphate binding. In the unbound state, Arg-303 forms a salt bridge with Glu-34, and in the liganded structure it interacts closely with the substrate C6-phosphate. The position of the sugar in the binding site would require a large movement prior to achieving the proper position for covalent catalysis with the Schiff base-forming Lys-229. The movement most likely involves a change in the location of the more loosely bound C6-phosphate. This result suggests that the substrate has one position in the Michaelis complex and another in the covalent complex. Such movement could trigger conformational changes in the carboxyl-terminal region, which has been implicated in substrate specificity.

Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis.

We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1, 4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P(3) binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P(3) binding, increased EC(50) values for Fru(1,6)P(2)-evoked release of bound Ins(1,4,5)P(3) and increased K(i) values for Ins(1,4, 5)P(3)-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P(3). Computer-simulated docking of Ins(1,4,5)P(3) in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P(3) to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P(2) binding. Results indicate that Ins(1,4,5)P(3) binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P(3) and Fru(1, 6)P(2) for binding. Although Ins(1,4,5)P(3) binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4, 5)P(3)-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P(3) in other proteins.

Identification of conserved promoter elements for aldB and isozyme specific residues in aldolase B.

The comparison of three complete aldolase B genes-including known and putative regulatory elements-is presented. The third aldolase B gene was provided by the complete aldB gene sequence (14803 bp) encoding the rabbit aldolase B isozyme. The promoter sequence alignment included the nonmammalian chicken aldolase B gene and confirms the promoter sequence conservation of those elements where trans-factor binding has been demonstrated in rat aldB. Moreover, the alignment reveals conserved sequences that may represent previously unidentified promoter elements that are present in all aldBs or specifically in the mammalian aldB promoters. One remarkable feature is a poly-purine segment found between the CAAT and TATA elements. In the mammalian promoters, this is exclusively a 9-10 bp poly-dA stretch. The avian promoter has an additional stretch of eight dG-bases immediately upstream of the poly-dA. Alignment of a portion of intron 1 of the chicken, human, and rabbit aldB genes reveals conserved sequences that are likely candidates for a reported positive activation sequence. In addition, the amino acid sequences of all eight known aldolase B isozymes is compared to the other vertebrate aldolases. A number of aldolase B-specific residues are identified that cluster in the carboxyl-portion of the sequence. With the exception of residue C268, these residues are not found near the active site, although, they are likely to be responsible for the substrate specificity of aldolase B.

Screening for hereditary fructose intolerance mutations by reverse dot-blot.

An assay is described which is useful for genetic screening of the two most prevalent mutations that cause hereditary fructose intolerance (HFI). Both mutations lie within exon 5 of the aldolase B gene. Amplification of exon 5 from genomic DNA isolated from peripheral lymphocytes using biotinylated aldolase B-specific primers yields a biotin-tagged probe. This probe is hybridized to complementary poly(dT)-tailed allele specific oligonucleotides (ASOs) that are bound to a nylon membrane. The length of the ASOs, the amount bound to the membrane and the time of hybridization are optimized for discrimination of all four alleles under the same hybridization conditions. Detection of biotinylated amplified DNA is performed by creating an avidin-alkaline phosphatase complex and visualization by chemiluminescence. This assay can rapidly detect the two mutations, A149P and A174D, which cause >70% of HFI worldwide, and offers a rapid and sensitive assay that is much less invasive for the diagnosis of this often difficult to diagnose disorder.

Identification of neuronal isozyme specific residues by comparison of goldfish aldolase C to other aldolases.

A 2061 bp cDNA encoding a goldfish (Carassius auratus) aldolase was isolated from a goldfish brain library. The deduced 362 amino acid sequence is more similar to vertebrate brain (aldolase C) and muscle aldolases (aldolase A) than to the liver isozymes (aldolase B). Northern blot analysis indicates strong expression of the mRNA in brain but not in liver or muscle, which indicates that this is aldolase C rather than aldolase A. Analysis of all known vertebrate aldolase amino acid sequences reveals five residues; Leu-57, Arg-314, Thr-324, Glu-332, and Gly-350 that are present exclusively in aldolase Cs. The goldfish clone possesses all five residues. The residues are primarily located in the carboxyl-terminal region of the enzyme and may play a role in determining the neuronal isozyme-specific properties of the enzyme. Furthermore, the existence of an aldolase C in a teleost fish has implications with respect to the timing of genome duplication events that are thought to have been critical in vertebrate evolution.

Antisense inhibition of R-cognin expression modulates differentiation of retinal neurons in vitro.

PURPOSE: Retina cognin (R-cognin) is a 50 kDa membrane-associated polypeptide expressed during retinogenesis where it is involved in mediating tissue-specific cell-cell interactions. In addition to its intercellular role in aggregation, R-cognin may act as a cell surface signaling molecule. An antisense oligonucleotide was used to inhibit R-cognin expression and to investigate the effects of this inhibition on subsequent neuronal differentiation. METHODS: Cultures of retina cells were prepared from 6 day (E6) and 8 day (E8) chicken embryos and were incubated with a deoxyoligonucleotide complimentary to 20 bases of the sequence encoding R-cognin or random oligonucleotides. The levels of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD), markers of cholinergic and GABAergic differentiation, respectively, were detected by Western blots on protein extracts from treated cultures. RESULTS: The antisense treatment inhibited ChAT levels at E6 and GAD levels at E8. The treatment resulted in no decrease in the level of the enzyme glyceraldehyde 3-phosphate dehydrogenase. A random oligonucleotide did not affect the levels of any of the proteins. CONCLUSIONS: These results confirm the cell recognition role of R-cognin and suggest that it is important in intracellular signaling cascades necessary for normal retina development.

Developmental localization of retina cognin synthesis by in situ hybridization.

Retina cognin (R-cognin) is a 50 kDa protein involved in cell recognition and neuronal differentiation during development of the embryonic chick retina. Initial characterization of a partial cDNA encoding R-cognin revealed a striking similarity to the cDNA encoding protein disulfide isomerase (PDI), a 57 kDa multifunctional protein. The exact nature of the relationship between R-cognin and PDI is not known; however, both proteins appear to be encoded by the same gene. In the present study, we developed cRNA probes to examine the expression of R-cognin and PDI transcripts in embryonic chick retina and liver. In the retina, the amount of transcript decreased with embryonic age, in parallel to a similar decrease in R-cognin protein. In the liver, where PDI is prominently expressed, the amount of transcript was not developmentally regulated. The spatial and temporal pattern of expression of the R-cognin-encoding retinal transcript was examined by in situ hybridization. R-cognin mRNA was expressed in cells across the retina early in retinogenesis, but became restricted to the cells of the inner retina later in development. This pattern of expression was the same as the developmental pattern of R-cognin protein [Dobi et al., Invest. Ophthalmol. Vis. Sci. 27, (1986) p. 323-329], thus, demonstrating that this secreted protein functions at the surface of the cells where it is transcribed.

Metabolic compartmentation in living cells: structural association of aldolase.

The glycolytic enzyme aldolase is concentrated in a domain around stress fibers in living Swiss 3T3 cells, but the mechanism by which aldolase is localized has not been revealed. We have recently identified a molecular binding site for F-actin on aldolase, and we hypothesized that this specific binding interaction, rather than a nonspecific mechanism, is responsible for localizing aldolase in vivo. In this report, we have used fluorescent analog cytochemistry of a site-directed mutant of aldolase to demonstrate that actin-binding activity localizes this molecule along stress fibers in quiescent cells and behind active ruffles in the leading edge of motile cells. The specific cytoskeletal association of aldolase could play a structural role in cytoplasm, and it may contribute to metabolic regulation, metabolic compartmentation, and/or cell motility. Functional duality may be a widespread feature among cytosolic enzymes.

Disruption of the aldolase A tetramer into catalytically active monomers.

The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Cross-linking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation. There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in many proteins.

The molecular nature of the F-actin binding activity of aldolase revealed with site-directed mutants.

We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg --> Ala), K107A (Lys --> Ala), and R148A (Arg --> Ala), had minimal actin binding activity relative to wild type (wt) aldolase, and one mutant, K229A (Lys --> Ala), had intermediate actin binding activity. A mutant with approximately 4,000-fold reduced catalytic activity, D33S (Asp --> Ser), had normal actin binding activity. The aldolase substrates and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and dihydroxyacetone phosphate, reversed the gelling of wt aldolase and F-actin, consistent with at least partial overlap of catalytic and actin-binding sites on aldolase. Molecular modeling reveals that the actin-binding residues we have identified are clustered in or around the catalytic pocket of the molecule. These data confirm that the aldolase-actin interaction is due to specific binding, and they suggest that electrostatic interactions between specific residues, rather than net charge, mediate this interaction. Low concentration of wt and D33S aldolase caused formation of high viscosity actin gel networks, while high concentrations of wt and D33S aldolase resulted in solation of the gel by bundling actin filaments, consistent with a potential role for this enzyme in the regulation of cytoplasmic structure.

A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation.

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate): D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13] was changed to arginine by site-directed mutagenesis. The kcat of the resulting mutant protein, K146R, was 500 times slower than wild-type in steady-state kinetic assays for both cleavage and condensation of fructose-1,6-bis(phosphate), while the K(m) for this substrate was unchanged. Analysis of the rate of formation of catalytic intermediates showed K146R was significantly different from the wild-type enzyme and other enzymes mutated at this site. Single-turnover experiments using acid precipitation to trap the Schiff base intermediate on the wild-type enzyme failed to show a build-up of this intermediate on K146R. However, K146R retained the ability to form the Schiff base intermediate as shown by the significant amounts of Schiff base intermediate trapped with NaBH4. In the single-turnover experiments it appeared that the Schiff base intermediate was converted to products more rapidly than it was produced. This suggested a maximal rate of Schiff base formation of 0.022 s-1, which was close to the value of kcat for this enzyme. This observation is strikingly different from the wild-type enzyme in which Schiff base formation is > 100 times faster than kcat. For K146R it appears that steps up to and including Schiff base formation are rate limiting for the catalytic reaction. The carbanion intermediate derived from either substrate or product, and the equilibrium concentrations of covalent enzyme-substrate intermediates, were much lower on K146R than on the wild-type enzyme. The greater bulk of the guanidino moiety may destabilize the covalent enzyme-substrate intermediates, thereby slowing the rate of Schiff base formation such that it becomes rate limiting. The K146R mutant enzyme is significantly more active than other enzymes mutated at this site, perhaps because it maintains a positively charged group at an essential position in the active site or perhaps the Arg functionally substitutes as a general acid/base catalyst in both Schiff base formation and in subsequent abstraction of the C4-hydroxyl proton.

Expression of aldolase A steady-state mRNA is delayed relative to other muscle-specific genes during differentiation of chicken myoblasts.

Expression of several muscle-specific genes was monitored during chicken muscle development and myoblast differentiation in primary cultures. The individual patterns of expression for many muscle-specific genes are well documented in ovo and in other model systems of muscle development. However, comparison of aldolase A to other muscle-specific genes in one system has not been reported. Both sarcomeric and cytosolic genes important for the adult muscle fiber were examined in order to elucidate their timing of expression and its relationship to cell fusion. Steady-state mRNA expression was measured using RNase protection assays with cRNA probes generated from cDNA clones for muscle creatine kinase, fast skeletal troponin-T, embryonic myosin heavy chain, and aldolase A. Nonmuscle genes expressed largely in the embryo, aldolase C and beta-actin, were used as controls. The expression of all six genes revealed differences in temporal expression patterns between limb and axial muscle. The temporal expression patterns of all six genes were also monitored in primary myoblast cultures relative to myoblast fusion. In both axial and limb myoblast cultures most of the muscle-specific genes were expressed prior to fusion. During the differentiation of myoblasts to myotubes there was a biphasic pattern in the expression of the muscle-specific genes. The appearance of measurable mRNA was detected by 16 hr in culture, prior to appreciable fusion of the cells. During further differentiation the expression increased gradually and then more rapidly at 96 hr, once fusion was complete. Meanwhile, the nonmuscle embryonic gene expression declined only slightly. For one gene, aldolase A, expression was delayed relative to the other muscle-specific genes, both in the appearance of measurable mRNA and in the later rapid increase in mRNA.

Noncoordinate changes in the steady-state mRNA expressed from aldolase A and aldolase C genes during differentiation of chicken myoblasts.

In chickens, as in all vertebrates, tissue-specific expression of aldolase isozymes A, B, and C is developmentally coordinated. These developmental transitions in aldolase expression have been studied most extensively by charting enzyme activity during normal and abnormal development of specific vertebrate tissues. Indeed, aldolase expression has been a key marker for normal differentiation and for retrodifferentiation during carcinogenesis. Aldolase expression during chicken myoblast differentiation offers a model for investigating the regulatory mechanisms of these developmental transitions at the level of gene expression. For these studies, cDNAs encoding the most isozyme-specific regions of both chicken aldolase A and C were cloned. The chicken aldolase A cDNA represents the first report of this sequence. Aldolase steady-state mRNA expression was measured during chicken myoblast differentiation in primary cultures using RNase protection assays with cRNA probes generated from these aldolase cDNA clones. Steady-state mRNA for aldolase C, the predominant embryonic aldolase isozyme in chickens, did not significantly change throughout myoblast differentiation. In contrast, expression of steady-state mRNA for aldolase A, the only aldolase isozyme found in adult-skeletal muscle, was not detected until after myoblast fusion was approximately 50% completed. Aldolase A expression gradually increased throughout myoblast differentiation until approximately 48 h after fusion was completed when there was a dramatic increase. These results are contrasted with those of Turner et al. (1974) [Dev Biol 37:63-89] that showed a coordinated switch in isozyme activities between the embryonic aldolase C and the muscle-specific aldolase A. This discordant expression indicates that the aldolase A and C genes may employ different regulatory mechanisms during myoblast differentiation.

Molecular basis of hereditary fructose intolerance: mutations and polymorphisms in the human aldolase B gene.

Mutations in the human aldolase B gene that result in hereditary fructose intolerance have been characterized extensively. Although the majority of subjects have been from northern Europe, subjects from other geographical regions and ethnic groups have been identified. At present 21 mutations have been reported; 15 of these are single base substitutions, resulting in nine amino acid replacements, four nonsense codons, and two putative splicing defects. Two large deletions, two four-base deletions, a single-base deletion, and a seven-base deletion/one-base insertion have been found. This last mutation leads to a defect in splicing and it is likely that one of the small deletions does as well. Regions of the enzyme where mutations have been observed recurrently are encoded by exons 5 and 9. Indeed, the three most common mutations are found in these exons. Two of these prevalent HFI mutations arose from a common ancestor and spread throughout the population by genetic drift. This finding was based on linkage to two sequence polymorphisms, which are among very few informative polymorphic markers that have been identified within the aldolase B gene. Because of the prevalence of a few HFI alleles, and the recent advances in molecular methods for identifying and screening for mutation, the diagnosis of HFI by molecular screening methods should become routine. These molecular diagnostic methods will be extremely beneficial for this often difficult to diagnose and sometimes fatal disease.

Characterization of recombinant human aldolase B and purification by metal chelate chromatography.

Recombinant human aldolase B and the native enzyme purified from human liver were found to be identical in size, charge, structure, Km constants for fructose-1,6-bis(phosphate) and fructose-1-phosphate, and the activity ratio of the two substrates. Thus recombinant aldolase B is a valid model for the native enzyme and can be used to study mutations that cause hereditary fructose intolerance or others designed in the active site. Addition of six histidine residues to the amino-terminus of the recombinant enzyme did not alter its structural or functional characteristics and allowed for purification by immobilized metal affinity chromatography. This purification protocol does not require a stable or active enzyme and will facilitate the study of mutant aldolase B enzymes that would otherwise be difficult to purify.

Lysine-146 of rabbit muscle aldolase is essential for cleavage and condensation of the C3-C4 bond of fructose 1,6-bis(phosphate).

Lysine-146 of rabbit muscle aldolase (D-fructose-1,6-biphosphate aldolase, EC 4.1.2.13) is absolutely conserved in class I (Schiff base) aldolases and has been implicated previously in catalysis by protein modification. Site-directed mutagenesis was used to change lysine-146 to alanine, glutamine, leucine, or histidine, creating the mutant enzymes K146A, K146Q, K146L, and K146H, respectively. These mutant proteins were expressed at high levels in bacteria and were purified by substrate affinity elution from CM-Sepharose, the same method that is used for the wild-type enzyme. The mutants K146A, K146Q, and K146L had substrate cleavage rates below standard detection levels. Modified cleavage assays indicated that these enzymes were (0.5-2) x 10(6)-fold decreased in the rate of catalysis of fructose 1,6-bis(phosphate) (Fru-1,6-Pa)cleavage. The K146H enzyme, however, was approximately 2000-fold slower than wild type in the rates of both cleavage and condensation of Fru-1,6-P2. In assays for the presence of enzymatic intermediates, all of the mutant enzymes were able to catalyze formation of the carbanion intermediate with dihydroxyacetone phosphate, whereas this intermediate was below the level of detection with Fru-1,6-P2. Single-turnover experiments with these enzymes in excess over radiolabeled Fru-1,6-P2 were used to measure the rates of Schiff base and product formation. The rate of Schiff base formation was decreased in each of the mutant enzymes, yet the magnitude of this decrease was less than the reduction in the respective kcat. These mutations had a much larger effect, however, on the rate of C3-C4 bond breaking, showing that Lys-146 is crucial at this step of the catalytic cycle.