The molecular architecture of the rabbit skeletal muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) tetramer has been determined to 2.7-A resolution. Solution of the three-dimensional structure of rabbit muscle aldolase utilized phase information from a single isomorphous Pt(CN)4-derivative, which was combined with iterative-phase refinement based upon the noncrystallographic 222-fold symmetry exhibited by the tetramer subunits. The electron-density map calculated from the refrned phases (mf = 0.72) was interpreted on the basis of the known amino acid sequence (363 amino acids per subunit). The molecular architecture of the aldolase subunit corresponds to a singly wound fl-barrel of the parallel a/fl class structures as has been observed in triose phosphate isomerase, pyruvate kinase, phosphogluconate aldolase, as well as others. Close contacts between tetramer subunits are virtually all between regions of hydrophobic residues. Contrary to other ,8-barrel structures, the known active-site residues are located in the center of the ,8-barrel and are accessible to substrate from the COOH side of the fl-barrel. Biochemical and crystallographic data suggest that the COOH-terminal region of aldolase covers the active-site pocket from the COOH side of the fl-barrel and mediates access to the active site. On the basis of sequence studies, active-site residues as well as residues lining the active-site pocket have been totally conserved throughout evolution. By comparison, homology in the COOH-terminal region is minimal. It is suggested that the amino acid sequence of the COOH-terminal region may be, in part, the basis for the variable specific activities aldolases exhibit toward their substrates.Aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) is an ubiquitous and abundant glycolytic enzyme that plays a central and pivotal role in glycolysis and fructose metabolism. Aldolases from all species catalyze the reversible aldol cleavage of fructose 1,6-bisphosphate (Fru-1,6-P2) into the triose phosphates, Dglyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis proceeds by two distinct chemical pathways in aldolases. In class I aldolases, found in plants and higher animals, catalysis depends upon Schiff-base formation with the substrate (1), whereas in class II aldolases, found mostly in molds and bacteria, catalysis requires a metal cofactor such as Zn2+ (2). Demonstrable activity by aldolases also exists toward substrates such as fructose 1-phosphate (Fru-1-P), and the differential activity by aldolases toward Fru-1,6-P2 and Fru-1-P has been used as a basis to discriminate between the various isozymes in vertebrates (3). In rabbit tissues, aldolase A has been isolated from muscle, aldolase B from liver, and aldolase C from brain. The three forms have been purified to homogeneity and extensively characterized (3-5). The enzymes have a relative molecular mass (Mr) of approximately 158,000 and a tertiary structure composed of...
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 3 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.Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a ubiquitous glycolytic enzyme that catalyzes the reversible cleavage of D-fructose 1,6-bisphosphate to D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, by an ordered uni-bi mechanism (1). Two classes of glycolytic aldolases can be distinguished that use different mechanisms to cleave the same substrate: Class I aldolases, present in higher eucaryotes and plants, catalyze their reaction mechanism via a Schiff base formation with a keto substrate whereas Class II aldolases, present in eubacteria and lower eucaryotes, require a divalent metal ion as a cofactor (2).The catalytic mechanism of Class I fructose-1,6-bisphosphate aldolase has been well established in terms of reaction mechanism intermediates (3), and the forward reaction can be represented by the following minimal reaction (Reaction 1) consistent with the kinetic data (4), in which a lysine residue on the enzyme forms covalent intermediates (5) with the keto substrates, Fru-P 2 , 1 Fru-P, and DHAP. In this forward reaction, Schiff base formation with Fru-P 2 , EϭDG, is thought to occur through transient formation of a dipolar carbinolamine-1 (4, 6) on the lysine nucleophile. Proton exchange with the dipolar carbinolamine results in the neutral species-2, which upon further protonation of the hydroxyl becomes dehydrated forming the protonated imine form of the Schiff base-3 (8). Proton abstraction of the Fru-P 2 O 4 hydroxyl initiates a rearrangement resulting in cleavage of the substrate C 3 -C 4 bond and enamine formation in the active si...
Mixed disulphide formation in the presence of oxidized glutathione reversibly inactivates rabbit skeletal muscle aldolase. Inactivation is allosteric, preferentially modifying Cys-72 on the surface of the aldolase homotetramer distant from active-site locations and subunit interfaces. Ion-exchange chromatography fractionates partly inactivated aldolase into three distinct enzymic species: unmodified enzyme, inactive fully modified enzyme corresponding to one thiol reacted per subunit, and inactive singly modified enzyme in which only one thiol has reacted. Acid-precipitable enzymic intermediates formed in the presence of substrate, D-fructose 1,6-bisphosphate, and product, dihydroxyacetone phosphate, indicates that active site binding is unaffected upon modification. The absence of enamine carbanion formation in the presence of substrate but not product is consistent with mixed disulphide formation's blocking -C-C- cleavage and/or subsequent D-glyceraldehyde 3-phosphate release. Inactivation upon single subunit modification and substrate protection against modification denotes that the blocked step is associated with a long-range conformational transition involving highly co-operative subunit behaviour.
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