Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates

The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine. Using active site mutants of FBPA I from Thermoproteus tenax, we have solved the crystal structures of the enzyme covalently bound to the carbinolamine of the substrate fructose 1,6-bisphosphate and noncovalently bound to the cyclic form of the substrate. The structures, determined at a resolution of 1.9 A and refined to crystallographic R factors of 0.148 and 0.149, respectively, represent the first view of any FBPA I in these two stages of the reaction pathway and allow detailed analysis of the roles of active site residues in catalysis. The active site geometry of the Tyr146Phe FBPA variant with the carbinolamine intermediate supports the notion that in the archaeal FBPA I Tyr146 is the proton donor catalyzing the conversion between the carbinolamine and Schiff base. Our structural analysis furthermore indicates that Glu187 is the proton donor in the eukaryotic FBPA I, whereas an aspartic acid, conserved in all FBPA I enzymes, is in a perfect position to be the general base facilitating carbon-carbon cleavage. The crystal structure of the Trp144Glu, Tyr146Phe double-mutant substrate complex represents the first example where the cyclic form of beta-fructose 1,6-bisphosphate is noncovalently bound to FBPA I. The structure thus allows for the first time the catalytic mechanism of ring opening to be unraveled.     

Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.     

Stereospecific proton transfer by a mobile catalyst in mammalian fructose-1,6-bisphosphate aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys(146) --> Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys(229) in native aldolase. Mutation of conserved active site residue Lys(146) to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr(363) hydroxyl within 2.9A of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr(363) is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys(146) positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr(363) interaction with Lys(146) and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.     

High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit

Crystal structures were determined to 1.8 A resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent alpha-helical regions that promote phosphate binding in the active site.     

Stereochemistry of nonnatural aldol reactions catalyzed by DHAP aldolases

A coupled enzymatic assay was developed for quantitative determination of the stereoisomeric products formed in aldol reactions catalyzed by dihydroxyacetone phosphate (DHAP)-dependent aldolases. Three of the four stereoisomers could be determined directly; the fourth one was calculated. This procedure is based on the reversibility of the aldol reaction and requires no derivatization or work-up of the product samples, only removal or inactivation of the biocatalyst. In comparison with other methods the enzymatic assay is highly accurate and fast. Determination of isomer formation with 10 different acceptor substrates applying this procedure gave unprecedented insight in the stereochemistry of fructose-1,6-bisphosphate aldolase from Staphylococcus carnosus and l-rhamnulose-1-phosphate aldolase from E. coli.     

A simple approach to detect active-site-directed enzyme-enzyme interactions. The aldolase/glycerol-phosphate-dehydrogenase enzyme system

A novel approach has been elaborated to identify the mechanism of intermediate transfer in interacting enzyme systems. The aldolase/glycerol-3-phosphate-dehydrogenase enzyme system was investigated since complex formation between these two enzymes had been demonstrated. The kinetics of dihydroxyacetone phosphate conversion catalyzed by the dehydrogenase in the absence and presence of aldolase was analyzed. It was found that the second-order rate constant (kcat/Km) of the enzymatic reaction decreases due to the formation of a heterologous complex. The decrease could be attributed to an increase of the Km value since kcat did not change in the presence of aldolase. In contrast, an apparent increase in the second-order rate constant of dihydroxyacetone phosphate conversion by the dehydrogenase was observed if the triose phosphate was produced by aldolase from fructose 1,6-bisphosphate (consecutive reaction). Moreover, no effect of dihydroxyacetone phosphate on the dissociation constant of the heterologous enzyme complex could be detected by physico-chemical methods. The results suggest that the endogenous dihydroxyacetone phosphate produced by aldolase complexed with dehydrogenase is more accessible for the dehydrogenase than the exogenous one, the binding of which is impeded due to steric hindrance by bound aldolase.     

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

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

Structure of a Class I Tagatose-1,6-bisphosphate Aldolase: INVESTIGATION INTO AN APPARENT LOSS OF STEREOSPECIFICITY*

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)₈ fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys²⁰⁵, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2–C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.     

A conserved glutamate residue exhibits multifunctional catalytic roles in D-fructose-1,6-bisphosphate aldolases.

The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.     

The proton exchange of the pro-S hydrogen atom at C-1 in dihydroxyacetone phosphate and D-fructose 1,6-bisphosphate catalysed by class-I and class-II aldolases.

The efficacy of class-I and class-II aldolases in catalysing the C-1 proton exchange in fructose 1,6-bisphosphate and dihydroxyacetone phosphate was investigated. The rate of this reaction was at least two orders of magnitude slower in class-II than in the class-I aldolases. It is suggested that this difference reflects the formation of different intermediates in the reactions catalysed by the two classes of aldolase.     

Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.     

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.     

Antibody inhibition of external aldolase activity in spinach chloroplast preparations

Abstract Antibodies (rabbit) have been prepared against total stroma from isolated spinach (Spinacia oleracea L. cv. Viking II) chloroplasts. These antibodies inhibited most of the aldolase activity present outside the chloroplasts in preparations of intact (80–95%) chloroplasts. They also reduced the amount of labelled fructose-1,6-bisphosphate found in the medium after ¹⁴CO₂ fixation with such preparations. Both intact and broken chloroplasts were strongly agglutinated by the antibodies. The results indicate that the external fructose-1,6-bisphosphate was formed from excreted dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase present outside the chloroplasts. The contamination of organelle preparations with free enzymes or enzymes adsorbed on the outer surface of the organelles is probably a general phenomenon. It is suggested that antibodies can be used as a tool to detect and selectively inhibit such contaminating enzyme activities.     

Structure of tagatose-1,6-bisphosphate aldolase. Insight into chiral discrimination, mechanism, and specificity of class II aldolases

Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate. The high resolution (1.45 A) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined. Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer. A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations. An unusual Na+ binding site is created using a pi interaction with Tyr183 in addition to five oxygen ligands. The catalytic Zn2+ is five-coordinate using three histidine nitrogens and two PGH oxygens. Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism. Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases. The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme. The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry.     

Interaction of the dissociable glycerol-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase. Quantitative analysis by an extrinsic fluorescence probe

Cytoplasmic sn-glycerol-3-phosphate dehydrogenase, labelled covalently with fluorescein isothiocyanate, shows an enzyme-concentration-dependent fluorescence anisotropy. The anisotropy versus enzyme concentration curve is shifted towards higher concentrations when substrates are present. The comparison of the dissociation constants estimated from anisotropy measurements and derived from kinetic experiments suggests that the substrate-induced dissociation of the dimeric dehydrogenase is slow with respect to the enzymatic reaction catalyzed by either its monomeric or dimeric form. The fluorescence anisotropy of the fluorescent dye-labelled dehydrogenase increase with time upon addition of unlabelled fructose-1,6-bisphosphate aldolase approaching a limiting value. This fact indicates the binding of fructose-1,6-bisphosphate aldolase aldose aldolase to glycerolphosphate dehydrogenase. A model is proposed assuming simultaneous binding of tetrameric fructose-1,6-bisphosphate aldolase to monomeric and dimeric glycerolphosphate dehydrogenase with 1:1 stoichiometry. The dissociation constants, as parameters fitted to the experimental curves, were estimated as 0.2 microM and 1 microM for aldolase-dimeric-glycerolphosphate-dehydrogenase and aldolase-monomeric-glycerolphosphate-dehydrogenase complexes respectively.     

Role of mono- and divalent metal cations in the catalysis by yeast aldolase

The rate of deuterium exchange between [1-(S)-2H]dihydroxyacetone 3-phosphate and the solvent catalyzed by native and metal-substituted yeast aldolases has been measured. In the presence of 0.1 M potassium acetate at 15 degrees C, pH 7.3, the deuterium exchange reaction catalyzed by native yeast aldolase has a kcat of 95 s-1. In contrast to the 7-fold activity enhancement by 0.1 M potassium ion (relative to 0.1 M sodium ion) of the cleavage of D-fructose 1,6-bisphosphate catalyzed by native yeast aldolase, a negligible (1.1-fold) activation by 0.1 M potassium ion is observed in the rate of dedeuteration of [1(S)-2H]dihydroxyacetone 3-phosphate. The order of reactivity of the yeast metalloaldolases in the deuterium exchange roughly parallels that seen in the fructose bisphosphate cleavage reaction. These findings suggest that the carbonyl groups of enzyme-bound D-fructose 1,6-bisphosphate and dihydroxyacetone phosphate are both polarized by the active site divalent metal cation. A mechanistic formulation consistent with the results of this and the previous paper is presented.     

Aldolase-catalyzed diketone phosphate formation from oxoaldehydes. NMR studies and metabolic significance.

NMR spectroscopy showed fructose-1,6-bisphosphate aldolase from rabbit muscle accepts as substrates, in lieu of glyceraldehyde 3-phosphate, the oxoaldehydes methylglyoxal and phenylglyoxal but not hydroxymethylglyoxal. The enzyme catalyzed an aldol condensation between the oxoaldehyde and dihydroxyacetone phosphate to form a monophosphorylated diketone and was inactivated in the process. Circumvention of this reaction, by metabolism of oxoaldehydes to hydroxy acids, may be a metabolic role for the glyoxalase enzyme system. Transketolase and transaldolase were found not to accept oxoaldehydes as substrates in place of glyceraldehyde 3-phosphate.     

Equilibrium partition studies of the interaction between aldolase and myofibrils

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

Aldolase--An Important Enzyme in Controlling the Ribulose 1,5-bisphosphate Regeneration Rate in Photosynthesis

This study was done to explore an enzymatic mechanism for thephotosynthetic carbon reduction cycle whereby the rate of synthesisof ribulose 1,5-bisphosphate (RuBP) could be changed while thelevels of intermediates other than 3-phosphoglycerate and RuBPwere kept constant. Chloroplast aldolase was purified to homogeneityfrom spinach leaves. When the enzyme was assayed in the directionof fructose 1,6-bisphosphate synthesis in the presence of theconcentrations of the substrates reported in vivo, the activitywas severely inhibited by physiological concentrations of RuBP.The aldolase reaction proceeded with a sequential mechanism.The K_m for dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphatewere 0.45 mM and 40 µM, respectively. The activity wascompetitively inhibited by RuBP with respect to dihydroxyacetonephosphate. The K^I was 0.78 mM. The maximum activity of aldolasein spinach leaves was calculated as 1,360µmol (mg Chl)^–1h^–1 An equation to express the reaction for the synthesisof fructose 1,6-bisphosphate by aldolase was constructed topredict the metabolic rate of this reaction in vivo. The calculationclearly showed that aldolase is an important enzyme in controllingthe rate of RuBP regeneration. (Received March 25, 1991; Accepted August 12, 1991)     

Structure of human brain fructose 1,6-(bis)phosphate aldolase: linking isozyme structure with function

Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 A resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.