A Conserved Glutamate Residue Exhibits Multifunctional Catalytic Roles in D-Fructose-1,6-bisphosphate Aldolases (original) (raw)
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Protein Science, 2004
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 glyceraldehyde-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 Å 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. Keywords: isozyme specificity; structural enzymology; protein-protein interactions; isozyme-specific residues; structure/function Fructose-1,6-(bis)phosphate aldolases are ubiquitous enzymes that catalyze the reversible cleavage of fructose-1,6-(bis)phosphate (Fru-1,6-P 2 ) and fructose 1-phosphate (Fru-1-P) to dihydroxy-acetone phosphate (DHAP) and either glyceraldehyde-3-phosphate (G3P) or glyceraldehyde, respectively. The mechanisms of these aldolases occur by two distinct chemical paths . Class I aldolases of animals and higher plants use covalent catalysis through a Schiff-base intermediate with ketose sugar substrates. Class II aldolases of most bacteria and fungi require a divalent metal cation as a cofactor. Among the class I enzymes found in mammals, there are three tissue-specific isozymes of aldolase that have similar molecular masses and catalytic mechanisms: aldolase A (expressed primarily in muscle and red blood cells), aldolase B (expressed primarily in liver,
Biochemistry, 1994
Lysine-146 of rabbit muscle aldolase (D-fructose-1 ,6-bisphosphate 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 a t 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 106-fold decreased in the rate of catalysis of fructose 1,6-bis(phosphate) (Fru-l,6-P2) 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
Journal of Biological Chemistry, 2003
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. Vertebrate fructose-1,6-bisphosphate aldolase is a ubiquitous tetrameric enzyme that catalyzes reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways (1). The enzyme catalyzes the reversible aldol cleavage of Fru 1,6-P 2
Biochemistry, 2001
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 Å 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.
Journal of Molecular Biology, 2002
Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the b5-a7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-2 H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that¯exibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK a to a value suitable for its role in proton abstraction. These results highlight the need for further studies of the dynamics of the enzyme in order to fully understand the complexities of loop closure and catalysis in this enzyme.
European Journal of Human Genetics, 1999
Hereditary fructose intolerance (HFI) is an autosomal recessive human disease that results from the deficiency of the hepatic aldolase isoenzyme. Affected individuals will succumb to the disease unless it is readily diagnosed and fructose eliminated from the diet. Simple and noninvasive diagnosis is now possible by direct DNA analysis that scans for known and unknown mutations. Using a combination of several PCR-based methods (restriction enzyme digestion, allele specific oligonucleotide hybridisation, single strand conformation analysis and direct sequencing) we identified a novel six-nucleotide deletion in exon 6 of the aldolase B gene (∆6ex6) that leads to the elimination of two amino acid residues (Leu182 and Val183) leaving the message inframe. The three-dimensional structural alterations induced in the enzyme by ∆6ex6 have been elucidated by molecular graphics analysis using the crystal structure of the rabbit muscle aldolase as reference model. These studies showed that the elimination of Leu182 and Val183 perturbs the correct orientation of adjacent catalytic residues such as Lys146 and Glu187.
Structural and functional analysis of aldolase B mutants related to hereditary fructose intolerance
FEBS Letters, 2002
Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired function of human liver aldolase (B isoform). 25 enzyme-impairing mutations have been identi¢ed in the aldolase B gene. We have studied the HFI-related mutant recombinant proteins W147R, A149P, A174D, L256P, N334K and v v6ex6 in relation to aldolase B function and structure using kinetic assays and molecular graphics analysis. We found that these mutations a¡ect aldolase B function by decreasing substrate a⁄nity, maximal velocity and/or enzyme stability. Finally, the functional and structural analyses of the non-natural mutant Q354E provide insight into the catalytic role of Arg 303 , whose natural mutants are associated to HFI.
Archives of Biochemistry and Biophysics, 1988
The amino acid sequence of fructose-1,6-bisphosphate aldolase from Drosoph,ila melanogaster was determined and was compared with those of five vertebrate aldolases on record. The four identical polypeptide chains of the insect enzyme, acetylated at the N-terminus and three residues shorter than the vertebrate chains, contain 360 amino acid residues. Of these 190 (or 53%) are identical in all six enzymes and in addition 33 positions (or 9%) are occupied by homologous residues. Comparison with the muscletype isoaldolases from man and rabbit and the liver-type isoaldolases from man, rat, and chicken indicates an average sequence identity of '70 and 63%, respectively. Thus, the insect and the vertebrate muscle aldolases are probably coded by orthologous genes. On this basis an average rate of evolution of 3.0 PAM per lo8 years is calculated, documenting an evolutional divergence slower than that of cytochrome c (4.2 PAM/lo8 years). The rate is also lower than that of the liver isoform (3.6 PAM/lo* years). Secondary structure prediction analysis for Drosophila aldolase suggests the occurrence of 11-12 helical segments and 8-9 P-strands. The conspicuous alternation of these structures in all six aldolases, especially in the C-terminal 200 residues, is consistant with the formation of an Lup-barrel supersecondary structure as documented for several other glycolytic enzymes.
1999
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 E-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 Å. 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.