Topography and conformational changes of fructose-1,6-bisphosphate aldolase.

Structure of aldolase, its interaction with nucleotides, the path of enzyme reaction and the scheme of range of conformational changes of this enzyme are presented. Retrospectives and perspectives of aldolase topography investigations are included.     

Suicide inactivation of fructose-1,6-bisphosphate aldolase

2-Keto-4,4,4-trifluorobutyl phosphate (HTFP) was prepared from 3,3,3-trifluoropropionic acid. HTFP acts as an irreversible inhibitor of rabbit muscle aldolase: the loss of activity was time dependent and the inactivation followed a pseudo-first-order process. Values of 1.4 mM for the dissociation constant and 2.3 X 10(-2) s-1 for the reaction rate constant were determined. The kinetic constants do not depend on the enzyme concentration. No effect of thiols on the inactivation rate was detected. Only 1-2 mol of fluoride ions was liberated per inactivated subunit, indicative of a low partition ratio. Dihydroxyacetone phosphate protected the enzyme against the inactivation in a competitive manner, and glyceraldehyde 3-phosphate protected as if it formed a condensation product with HTPF. 5,5'-Dithiobis(2-nitrobenzoic acid) thiol titration showed the loss of one very reactive thiol group per enzyme subunit after inactivation. All those observations seem to agree with a suicide substrate inactivation of aldolase by HTPF.     

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.     

Rapid purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts

A rapid procedure for the purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts is presented which involves two steps; precipitation of bulk protein with polyethylene glycol and partitioning of remaining soluble protein in aqueous two-phase systems. A 94% pure preparation is obtained within 6h with a yield of 19%. A marked difference in the partition behaviour of the aldolase activity from whole leaf tissue suggested that the procedure is less efficient when leaf extract is used as starting material.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - PEG Polyethylene Glycol - SDS Sodium Dodecyl Sulfate     

Action of cathepsin D on fructose-1,6-bisphosphate aldolase.

Cathepsin D inactivated aldolase at pH values between 4.2 and 5.2; the chloride, sulphate or iodide, but not citrate or acetate, salts of sodium or potassium accelerated the rate of inactivation. Cathepsin D cleaved numerous peptide bonds in the C-terminus of aldolase, but the major site of cleavage in this region was Leu354-Phe355. The most prominent peptide products of hydrolysis were Phe-Ile-Ser-Asn-His-Ala-Tyr and Phe-Ile-Ser-Asn-His. Up to 20 amino acids were removed from the C-terminus of aldolase, but no further degradation of native aldolase was observed. By contrast, extensive degradation of the 40 000-Mr subunit was observed after aldolase was denatured. The cathepsin D-inactivated aldolase cross-reacted with antibodies prepared against native aldolase and had the same thermodynamic stability as native aldolase, demonstrated by differential scanning calorimetry and fluorescence quenching of tryptophan residues. Furthermore, the cathepsin-modified and native forms of aldolase were both resistant to extensive proteolysis by other purified cellular proteinases and lysosomal extracts at pH values of 4.8-8.0.     

Spatial clustering of isozyme-specific residues reveals unlikely determinants of isozyme specificity in fructose-1,6-bisphosphate aldolase

Vertebrate fructose-1,6-bisphosphate aldolase exists as three isozymes (A, B, and C) that demonstrate kinetic properties that are consistent with their physiological role and tissue-specific expression. The isozymes demonstrate specific substrate cleavage efficiencies along with differences in the ability to interact with other proteins; however, it is unknown how these differences are conferred. An alignment of 21 known vertebrate aldolase sequences was used to identify all of the amino acids that are specific to each isozyme, or isozyme-specific residues (ISRs). The location of ISRs on the tertiary and quaternary structures of aldolase reveals that ISRs are found largely on the surface (24 out of 27) and are all outside of hydrogen bonding distance to any active site residue. Moreover, ISRs cluster into two patches on the surface of aldolase with one of these patches, the terminal surface patch, overlapping with the actin-binding site of aldolase A and overlapping an area of higher than average temperature factors derived from the x-ray crystal structures of the isozymes. The other patch, the distal surface patch, comprises an area with a different electrostatic surface potential when comparing isozymes. Despite their location distal to the active site, swapping ISRs between aldolase A and B by multiple site mutagenesis on recombinant expression plasmids is sufficient to convert the kinetic properties of aldolase A to those of aldolase B. This implies that ISRs influence catalysis via changes that alter the structure of the active site from a distance or via changes that alter the interaction of the mobile C-terminal portion with the active site. The methods used in the identification and analysis of ISRs discussed here can be applied to other protein families to reveal functionally relevant residue clusters not accessible by conventional primary sequence alignment methods.     

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.     

MJ0400 from Methanocaldococcus jannaschii exhibits fructose-1,6-bisphosphate aldolase activity

The central carbon metabolism is well investigated in bacteria, but this is not the case for archaea. MJ0400-His₆ from Methanocaldococcus jannaschii catalyzes the cleavage of fructose-1,6-bisphosphate (FBP) to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate with a V _max of 33 mU mg⁻¹ and a K _m of 430 μM at 50 °C. MJ0400-His₆ is inhibited competitively by erythrose-4-phosphate with a K _i of 380 μM and displays heat stability with a half-life of c . 1 h at 100 °C. Hence, MJ0400 is the second gene encoding for an FBP aldolase in M. jannaschii . Previously, MJ0400 was shown to act as an 2-amino-3,7-dideoxy- d - threo -hept-6-ulosonic acid synthase. This indicates that MJ0400 is involved in both the carbon metabolism and the shikimate pathway in M. jannaschii .     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Crystallization and preliminary X-ray analysis of class II fructose-1,6-bisphosphate aldolase from Thermus caldophilus

In this study, we have crystallized class II fructose-1,6-bisphosphate aldolase (FBA) from Thermus caldophilus (Tca). Purified Tca FBA is a tetrameric enzyme of 305 residues, which crystallizes in the space group P2(1)2(1)2(1) (cell dimensions a = 98.9, b = 113.1, c = 115.7 A), with four molecules in the asymmetric unit. A complete diffraction data set was obtained from orthorhombic crystals at resolution of 2.2 A.     

Inactivation of fructose-1,6-bisphosphate aldolase by cathepsin L stimulation by ATP

Cathepsin L was capable of destroying rabbit muscle aldolase (d-fructose-1,6-bisphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) activity towards the substrate fructase 1,6-bisphosphate. The rate of loss of activity towards this substrate was stimulated (approx. 2-fold) by physiological concentrations of ATP and to a lesser degree by GTP, CTP, UTP, ADP and cyclic AMP, while PP_i and P_i decreased the rate of inactivation. Other proteinases (cathepsin B, cathepsin D, trypsin and chymotrypsin) also decreased aldolase activity toward fructose 1,6-bisphosphate more rapidly in the presence of ATP and more slowly in the presence of P_i. Cathepsin L, at higher concentrations, was capable of inactivating aldolase activity towards fructose 1-phosphate and extensively degrading the enzyme; these reactions were not affected by ATP and P_i. The thermostability of aldolase was also unaffected by these ligands. ATP and P_i had no effect on the rates of hydrolysis of other proteins (hemoglobin, bovine serum albumin, casein and azocasein) by cathepsin L. These data indicate that the effects of ATP and P_i was due to interactions of these ligands with aldolase that make the enzyme more vulnerable to limited but not extensive proteolysis; these ligands do not directly affect cathepsin L activity.     

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.     

Chemical trapping of complexes of dihydroxyacetone phosphate with muscle fructose-1,6-bisphosphate aldolase

Dihydroxyacetone phosphate (DHAP) in equilibrium with FDP aldolase of muscle is present in the form of two major covalent complexes. One, representing approximately 60% of total bound substrate, decomposes to Pi and methylglyoxal upon acid denaturation of the enzyme as first reported by Grazi and Trombetta [Grazi, E., & Trombetta, G. (1979) Biochem. J. 175, 361-365]. This is now shown to be the enzyme-eneamine phosphate reaction intermediate since Pi formation is prevented if the acid denaturation is done in the presence of potassium ferricyanide, an oxidant of the eneamine. The enzyme-eneamine aldehyde X Pi 6, presumed to be an intermediate of the slow methylglyoxal synthetase reaction of aldolase, must not be a significant source of the Pi produced upon denaturation and is probably not a significant component of the equilibrium. The oxidation product, the enzyme-imine of phosphopyruvaldehyde, is sufficiently stable in 1 N HCl, t1/2 = 76 min at 0 degree C, to be isolated with the trichloroacetic acid precipitated protein. A second covalent complex, approximately 20-24% of bound dihydroxyacetone [32P]phosphate, remains with the protein during acid denaturation and centrifugation. This acid-stable complex is formed rapidly and is chased rapidly by unlabeled substrate. Its stability in 1 N HCl is similar to that of the ferricyanide-oxidized derivative mentioned above. From this and its reactivity with cyanoborohydride in acid, this complex is thought to be the imine adduct of DHAP with aldolase 4 and/or the carbinolamine complex 3 present in the initial equilibrium. D-Glyceraldehyde 3-phosphate in the carbonyl form also forms an acid-precipitable complex with aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase.

We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A, c = 290.6 A and are suitable for a detailed structural analysis. The space group has been identified as P6(1)22 or enantiomorph. Data sets to approximately 2.9 A resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S. Daresbury. Diffraction beyond 2.5 A has been observed when large freshly grown crystals are used with the synchrotron beam. A data set to this resolution has been collected. Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.     

A Class II fructose-1,6-bisphosphate aldolase from a halophilic archaebacterium Haloferax mediterranei

Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) was purified 97-fold from a halophilic archaebacterium Haloferax mediterranei, with a specific activity of 2.8. The enzyme was characterized as a Class II aldolase on the basis of its inhibition by EDTA and other metal chelators. The enzyme had a specific requirement for divalent metal Fe(2+) for activity. Sulfhydryl compounds enhanced aldolase activity.     

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.     

Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii

The Calvin-Benson cycle fixes carbon dioxide into organic triosephosphates through the collective action of eleven conserved enzymes. Regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco-mediated carboxylation, requires two lyase reactions catalyzed by fructose-1,6-bisphosphate aldolase (FBA). While cytoplasmic FBA has been extensively studied in non-photosynthetic organisms, functional and structural details are limited for chloroplast FBA encoded by oxygenic phototrophs. Here we determined the crystal structure of plastidial FBA from the unicellular green alga Chlamydomonas reinhardtii (Cr). We confirm that CrFBA folds as a TIM barrel, describe its catalytic pocket and homo-tetrameric state. Multiple sequence profiling classified the photosynthetic paralogs of FBA in a distinct group from non-photosynthetic paralogs. We mapped the sites of thiol- and phospho-based post-translational modifications known from photosynthetic organisms and predict their effects on enzyme catalysis.     

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.     

Properties of fructose-1,6-diphosphate phosphatase and fructose-1,6-diphosphate aldolase fromPseudomonas putida

The fructose-1,6-P₂ (FDP) phosphatase, (FDPase) and FDP aldolase fromPseudomonas putida were partially purified by a combination of (NH₄)₂SO₄ fractionation and DEAE-Sephadex column chromatography. Michaelis-Menten kinetics were observed with, respect to FDP in both FDPase and FDP aldolase. TheK _m for FDP at pH 8.0 was 1.2×10⁻⁵M for FDPase and 3.0×10⁻⁵M for FDP aldolase. The specific activities of these two enzymes (assayed under optimal conditions in cell-free extracts ofP. putida grown ond-fructose), as well as their kinetic properties, are consistent with the suggestion that during growth ond-fructose most, of the FDP generated is converted to fructose-6-P (F-6-P), which is subsequently utilized via the Entner-Doudoroff pathway (EDP).     

The crystal structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster at 2.5 A resolution

The structure of fructose-1,6-bisphosphate aldolase from Drosophila melanogaster has been determined by X-ray diffraction at 2.5 A resolution. The insect enzyme crystallizes in space group P2(1)2(1)2(1) with lattice replacement with rabbit muscle aldolase as a search model has been employed to solve the structure. To improve the initial phases real space averaging, including phase extension from 4.0 to 2.5 A, has been applied. Refinement of the atomic positions by molecular dynamics resulted in a crystallographic R-factor of 0.214. The tertiary structure resembles in most parts that of the vertebrate aldolase from rabbit muscle. Significant differences were found in surface loops and the N- and C-terminal regions of the protein. Here we present the first aldolase structure where the functionally important C-terminal arm is described completely.