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.     

Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.     

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.     

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.     

Opposite changes with age in liver and muscle in the mitochondrial and soluble glucose-1,6-bisphosphate and 6-phosphogluconate dehydrogenase

Glucose-1,6-bisphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was markedly decreased in liver of adult rats (2 months of age) as compared to young rats (1-2 weeks of age). This regulator was found to be present in both the mitochondrial and soluble fractions of liver. Its concentration in both these fractions was decreased with age. Concomitant to the decrease in Glc-1,6-P2, which is a potent inhibitor of 6-phosphogluconate dehydrogenase, the activity of this enzyme was markedly increased with age in both the mitochondrial and soluble fractions. However, the increase in this enzyme's activity was more pronounced in the mitochondrial fraction. The mitochondrial enzyme was more susceptible to inhibition by Glc-1,6-P2 as compared to the soluble enzyme, and this may explain the greater enhancement in its activity with age in this fraction. The tibialis anterior muscle exhibited changes with age opposite to those found in liver; Glc-1,6-P2 concentration, in both the mitochondrial and soluble fractions of muscle increased with age, and this increase was accompanied by a concomitant reduction in the activity of the mitochondrial and soluble 6-phosphogluconate dehydrogenase. Similar to liver, the mitochondrial enzyme was more affected by age, as it also exhibited a greater susceptibility to inhibition by Glc-1,6-P2.     

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.     

Investigation of the relationship between tyrosyl residues and the adenosine 5'-monophosphate binding site of rabbit liver fructose-1,6-biphosphatase as studied by chemical modification and nuclear magnetic resonance spectroscopy

The effects of AMP, fructose 6-phosphate (Fru-6-P), fructose 2,6-bisphosphate (Fru-2,6-P2), and paramagnetic ions on the aromatic region of the proton nuclear magnetic resonance (NMR) spectrum of rabbit liver fructose-1,6-bisphosphatase have been investigated at 300 MHz. Two well resolved peaks in this region of the NMR spectrum are assigned to the protons from the aromatic ring of a tyrosyl residue of the enzyme by chemical modification with tetranitromethane and by nuclear Overhauser effects. Nitration of the tyrosyl residue causes desensitization of the enzyme to AMP inhibition as well as the loss of activity. In the presence of AMP during the modifications, 1 tyrosyl residue could be protected, presumably the one observed by NMR. Binding of AMP, an allosteric inhibitor of the enzyme, to rabbit liver fructose-1,6-bisphosphatase leads to an upfield shift of the tyrosyl proton signals in the NMR spectrum. No chemical shift or line broadening could be detected in the presence of the paramagnetic manganous ion, Fru-2,6-P2, or Fru-6-P. The negative intramolecular nuclear Overhauser effect from the ribose H2' proton to the adenine H8 proton of AMP suggested that AMP binds to the enzyme with an anti conformation about the glycosidic bond. The failure to observe intermolecular nuclear Overhauser effects between the tyrosyl residue and the protons of AMP indicates that the distances between them are greater than 4 A. On the basis of these observations, it is suggested that the AMP-related tyrosyl residue may be close to the AMP binding site, but it is not directly involved in ligand binding. Rather, the protection of this tyrosyl residue by AMP as observed by chemical modification experiments may well be due to a conformational change that results from covalent modification of the enzyme.     

Role of inosine 5'-phosphate in activating glucose-bisphosphatase

Glucose-bisphosphate (G1c-1,6-P2) phosphatase has been purified greater than 200-fold from the cytosol of mouse brain. As reported earlier, the enzyme requires inosine monophosphate (IMP) and Mg2+ for activity [Guha, S.K., & Rose, Z. B. (1982) J. Biol. Chem. 257, 6634-6637]. Kinetic parameters and the role of IMP have been further investigated. When Glc-1,6-P2 and IMP are both varied, double-reciprocal plots of the data form a parallel line pattern. With 2 mM Mg2+, the Km obtained for G1c-1,6-P2 is 20 microM and the Ka for IMP is 9 microM. Co2+, Mn2+, and Ni2+ activate less effectively than Mg2+. The apparent Ka for Mg2+ decreases with increasing G1c-1,6-P2, and the observed Km of G1c-1,6-P2 decreases with increasing Mg2+. The extrapolated value of the Ka of Mg2+ at infinite substrate is 86 microM. Mg2+ does not affect the Ka of IMP. The phosphatase activity is optimal at pH 7. The phosphatase is not completely specific since mannose 1,6-bisphosphate is hydrolyzed and guanosine monophosphate activates. However, fructose 1,6-bisphosphate is no more than a poor inhibitor, and adenine nucleotides are neither activators nor inhibitors. The products of the reaction are glucose-1-P and glucose-6-P, in a ratio of 2:3, and Pi. Both glucose-P's are competitive inhibitors with respect to IMP [Ki(glucose-1-P) = 5 microM; Ki(glucose-6-P) = 18 microM]. Neither glucose-P competes with G1c-1,6-P2. The demonstration of an exchange reaction between G1c-1,6-P2 and glucose-6-P is evidence for the phosphorylation of the enzyme by the substrate. The exchange reaction requires Mg2+ and is inhibited by IMP. The observation of the exchange reaction and its elimination by IMP indicates that the low level of phosphoglucomutase activity that remains with the phosphatase throughout purification is an inherent property of the phosphatase. The requirement of glucose-bisphosphatase for the nucleotide IMP is consistent with possible roles for both G1c-1,6-P2 and IMP in the control of the ATP level in the brain.     

Structure-based Reevaluation of the Mechanism of Class I Fructose-1,6-bisphosphate Aldolase

The enzymatic reaction carried out by class I fructose-1,6-bisphosphate aldolase is known in great detail in terms of reaction intermediates, but the precise role of individual amino acids in the active site is poorly understood. Therefore, on the basis of the crystallographic structure of the complex between aldolase and dihydroxyacetone phosphate a molecular modelling study was undertaken to predict the Michaelis complex with fructose-1,6-bisphosphate and several covalent enzymatic reaction intermediates. This model reveals the unknown 6-phosphate binding site and assigns distinct roles to crucial residues. Asp33 is responsible for aligning the 2-keto function of the substrate correctly for nucleophilic attack by Lys229, and plays a role in carbinolamine formation. Lys146 assists in carbinolamine dehydration and is essential for stabilising the developing negative charge on O4 of fructose-1,6-bisphosphate during hydroxyl proton abstraction by Glu187. Subsequently, Glu187 is also responsible for protonating C1 of the dihydroxyacetone phosphate enamine. In addition, the absolute configuration of the fructose-1,6-bisphosphate carbinol intermediate is shown to be (2S), in agreement with the crystal structure, but opposite from the interpretation in the literature of the stereospecific reduction of the aldolase fructose-1,6-bisphosphate complex with sodium borohydride. It is demonstrated that the outcome of the latter type of experiment critically depends on conformational changes triggered by Schiff base formation. Electronic Supplementary Material available.     

Rapid-quench and isotope-trapping studies on fructose-1,6-bisphosphatase

Rapid-quench kinetic measurements yielded presteady-state rate data for rabbit liver fructose-1,6-bisphosphatase (FBPase) (a tetramer of four identical subunits) that are triphasic: the rapid release of Pi (complete within 5 ms), followed by a second reaction phase liberating additional Pi that completes the initial turnover of two or four subunits of the enzyme (requiring 100-150 ms), and a steady-state rate whose magnitude depends on the [alpha-Fru-1,6-P2]/[FBPase] ratio. With Mg2+ in the presence of excess alpha-fructose 1,6-bisphosphate (alpha-Fru-1,6-P2) all four subunits turn over in the pre steady state; with Mn2+ only two of the four are active. Thus the expression of half-site reactivity is a consequence of the nature of the metal ion and not a subunit asymmetry. In the presence of limiting alpha-anomer concentrations only two of the four subunits now remain active with Mg2+ as well as with Mn2+ in the pre steady state. However, so that the amount of Pi released can be accounted for, a beta leads to alpha anomerization or direct beta utilization is required at the active site of one subunit. Such behavior is consistent with the two-state conformational hysteresis displayed by the enzyme and altered affinities manifested within these states for alpha and beta substrate analogues. Under these limiting conditions the subsequent steady-state rate is limited by the beta leads to alpha solution anomerization. These data in combination with pulse--chase experiments permit evaluation of the internal equilibrium, which in the case of Mg2+ is unequivocally higher in favor of product complexes and represents a departure from balanced internal substrate-product complexes.     

Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.     

2',5' A synthetase: allosteric activation by fructose 1,6-bisphosphate

Fructose 1,6-bisphosphate (fru-1,6-P2), but not other glycolytic intermediates, activates highly purified 2',5' A synthetases from rabbit reticulocyte lysates and from 2',5'-ADP-agarose purified extracts of interferon-treated HeLa cells without the addition of dsRNA. The 2',5' A was structurally and biologically identical to authentic 2',5' A. Micrococcal nuclease inhibited the activation of 2',5' A synthetase by poly(I)-poly(C), but did not affect activation by fru-1,6-P2. Addition of fru-1,6-P2 aldolase prevented the activation of 2',5' A synthetase by fru-1,6-P2.     

The significance of sedoheptulose 1,7-bisphosphate in the metabolism and regulation of the pentose pathway in liver

Rat liver cytosolic enzyme preparation catalyses the formation of sedoheptulose 1,7-P2 (60% of total heptulose-P formed) from hexose 6-P and triose 3-P (reverse mode of pentose pathway operation). Smaller amounts of sedoheptulose 1,7-P2 are also formed from ribose 5-P during the non-oxidative synthesis of hexose 6-P (forward pentose pathway operation). The apparent absence of erythrose 4-P in biological systems may be explained by its contribution to carbons 4,5,6 and 7 of sedoheptulose 1,7-P2 as well as its pronounced ability to exist in dimeric form. Apart from the aldolase catalyzed formation of sedoheptulose 1,7-P2, 6-phosphofructokinase also catalyses its formation from sedoheptulose 7-P and fructose 1,6-bisphosphatase catalyses its dephosphorylation. These three enzymes may contribute to the regulation of carbon flux through the near equilibrium reactions of the non-oxidative pentose phosphate pathway in vivo. The phosphotransferase enzyme of the L-type pentose pathway is also able to catalyse the interconversion of sedoheptulose mono and bisphosphates via D-glycero D-ido octulose-P.     

The synthesis of mannose 1-phosphate in brain

The interconversion of mannose-6-P and mannose-1-P in brain has been shown to be catalyzed by a distinct enzyme. The enzyme has been separated from most of the phosphoglucomutase activity of the brain. The residual phosphoglucomutase activity (less than 1%) may be associated with phosphomannomutase itself. Mannose-1,6-P2 or glucose-1,6-P2 is required for the reaction as well as a divalent cation (Mg2+ greater than Co2+ greater than Ni2+ greater than Mn2+). Glucose-1-P, glucose-6-P, and 2-deoxyglucose-6-P are also substrates or inhibitors. Other phosphorylated sugars tested, glucosamine-6-P, N-acetylglucosamine-6-P, galactose-6-P, fructose-6-P, ribose-5-P, and arabinose-5-P, do not affect the rate of the reaction when assayed in the presence of mannose-6-32P.     

Purification and properties of rabbit heart muscle aldolase.

Fructose diphosphate aldolase (D-fructose-1,6-biphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit heart has been purified and obtained in crystalline form. The preparations are homogeneous on the basis of disc gel electrophoresis and ultracentrifugation. The catalytic and the molecular properties indicate that this is aldolase A. A comparison was made between rabbit heart aldolase and the rabbit muscle enzyme. The sedimentation coefficient, energy of activation and Michaelis constant for Fru-1,6-P2 were found to be identical with the values obtained for the muscle enzyme. As in case of the muscle enzyme, heart aldolase was found to have a broad pH optimum, remarkable stability over a wide pH range, and the ability to form a Schiff base intermediate with dihydroxyacetone phosphate upon reduction with borohydride. Cleavage of the methionyl bonds with CNBr yields the same pattern as obtained with the muscle enzyme.     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

Complexes of muscle aldolase in equilibrium with fructose 1,6-bisphosphate

Minimum values for the content of covalent intermediates in the equilibria of muscle aldolase with its cleavable substrates have been determined by acid denaturation/precipitation. Ribulose 1,5-bisphosphate, a nonsubstrate that binds well to aldolase in the native state, does not form a covalent complex that is acid precipitable. The insoluble protein complexes with substrates fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, representing approximately 50% and approximately 60% of total bound substrate, are much more stable in acid and alkali than that with substrate 5-deoxyfructose 1,6-bisphosphate, suggesting that they have the form of protein-bound N-glycosides. Whether such complexes exist on the enzyme in the native state in addition to being formed subsequent to denaturation is unresolved. Both the acid-precipitable and nonprecipitable forms of fructose 1,6-bisphosphate are converted to triose phosphate products at the same rate, providing no kinetic evidence for a pool that is not on the main reaction path. Total fructose 1,6-bisphosphate liganded to enzyme returns to the free solution about 9 times for each net cleavage reaction. It is still not clear whether this is limited by the cleavage step or by release of glyceraldehyde phosphate.     

Evidence that aldolase and D-arabinose 5-phosphate are components of pentose pathway reactions in liver in vitro

An immunochemical procedure involving the reaction of liver aldolase antibody and rat liver enzyme preparation shows that conversion of ribose 5-P to hexose 6-P by reactions of the non-oxidative pentose pathway fails to occur in the absence of aldolase activity. Radioautography of pentose pathway products formed by liver enzyme catalysis of [U-14C] arabinose 5-P and unlabelled ribose 5-P illustrates the incorporation of 14C into ketopentose, sedoheptulose, fructose and glucose phosphates. There is approximate congruity of the mole specific radioactivity of the pentose and hexose phosphates. These findings are consistent with the proposal that L-pentose pathway reactions constitute the non-oxidative segment of the pathway in liver.     

Fructose-1,6-biphosphatase of the small intestine. Purification and comparison with liver and muscle fructose-1,6-bisphosphatases.

The occurrence of specific fructose-1,6-bisphosphatase [D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11] (Fru-1,6-P2ase) in the small intestine was confirmed. 1. Fru-1,6-P2ase was isolated from mouse small intestine by a simple method. The isolated enzyme preparation was an electrophoretically homogeneous protein. 2. The molecular weight and subunit molecular weight were 140,000 and 38,000, respectively. 3. The intestinal enzyme was electrophoretically distinct from the liver enzyme. 4. The kinetic properties of the purified intestinal enzyme were compared with those of the mouse liver and muscle enzymes. 5. Mouse intestinal and muscle Fru-1,6-P2ases hydrolyzed ribulose-1,5-bisphosphate in addition to fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate.