Isomer activation controls stereospecificity of class I fructose-1,6-bisphosphate aldolases

Abstract

Fructose-1,6-bisphosphate (FBP) aldolase, a glycolytic enzyme, catalyzes the reversible and stereospecific aldol addition of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (d-G3P) by an unresolved mechanism. To afford insight into the molecular determinants of FBP aldolase stereospecificity during aldol addition, a key ternary complex formed by DHAP and d-G3P, comprising 2% of the equilibrium population at physiological pH, was cryotrapped in the active site of Toxoplasma gondii aldolase crystals to high resolution. The growth of T. gondii aldolase crystals in acidic conditions enabled trapping of the ternary complex as a dominant population. The obligate 3(S)-4(R) stereochemistry at the nascent C3-C4 bond of FBP requires a si-face attack by the covalent DHAP nucleophile on the d-G3P aldehyde si-face in the active site. The cis-isomer of the d-G3P aldehyde, representing the dominant population trapped in the ternary complex, would lead to re-face attack on the aldehyde and yield tagatose 1,6-bisphosphate, a competitive inhibitor of the enzyme. We propose that unhindered rotational isomerization by the d-G3P aldehyde moiety in the ternary complex generates the active trans-isomer competent for carbonyl bond activation by active-site residues, thereby enabling si-face attack by the DHAP enamine. C-C bond formation by the cis-isomer is suppressed by hydrogen bonding of the cis-aldehyde carbonyl with the DHAP enamine phosphate dianion through a tetrahedrally coordinated water molecule. The active site geometry further suppresses C-C bond formation with the l-G3P enantiomer of d-G3P. Understanding C-C formation is of fundamental importance in biological reactions and has considerable relevance to biosynthetic reactions in organic chemistry.

Keywords: crystal structure; enzyme catalysis; enzyme mechanism; glycolysis; stereoselectivity.

Scheme 1.

Intermediates of the catalytic mechanism in class I aldolases.

Figure 1.

Progress of the aldol addition reaction in a class I aldolase. A, overall view of the subunit globular TIM-barrel structure (helices in red; sheets in yellow) and of the active-site pocket in TgALD (structure shown corresponds to 30-min soaking with FBP in crystallization buffer). In the 30-min structure, FBP is covalently bound to the Schiff base–forming Lys-231. Key active-site residues that participate in hydrogen bonding (gray dashes) with substrate and in catalytic activity are depicted in the close-up (right inset). Noteworthy is the hydrogen bond made between FBP O4 and Glu-189 and that with Lys-146. The FBP phosphates (P1 and P6) participate in extensive contacts with adjacent residues (some omitted). Residue numbering is shown for TgALD. B, crystal structures obtained corresponding to different soaking times of native TgALD crystals with FBP in the crystallization buffer and the resultant intermediates that were trapped at the incremental time points by flash-freezing soaked crystals in liquid nitrogen. Difference electron density maps were calculated from simulated-annealing F_o − F_c omit maps encompassing the substrate-binding site and are contoured at a 2.1σ level. Where appropriate, the covalently bound intermediates, DHAP-enamine (green) or FBP-Schiff base (pink), are modeled into the active site as well as the aldehyde triose-P product d-G3P (wheat). Water molecules (blue spheres) were also modeled where appropriate. Ligands juxtaposed with water molecules at the 0.5-min structure correspond to partially occupied configurations. Soaking for at least 10 min is required for FBP to fully populate the TgALD active site.

Figure 2.

pH activity profile of the FBP cleavage reaction for TgALD. Saturating concentrations of FBP were used to assay activity at pH 4–9 for every 0.5 pH unit. A double pK_a curve was fitted against the kinetic data using Equation 6 (see “Experimental procedures”) in GraFit (version 6.0.12). The acidic limb of the profile has a pK_a of 5.0, consistent with the pK_a of a glutamate residue. Error bars, S.E. (n ≥ 3 for each point).

Figure 3.

Reaction geometry associated with aldol addition implicating putative cis-trans rotational isomerization. A, active-site difference density calculated from a simulated-annealing F_o − F_c omit map is illustrated encompassing the substrate-binding site surrounding the cis-d-G3P aldehyde group in the 1-min structure and contoured at a 2.1σ level. Unambiguous modeling of the cis-configuration was possible. Refinement with the trans-configuration afforded a poor fit, as shown by the residual difference density (green) calculated from a F_o − F_c map (contour at 2.1σ). The corresponding 2F_o − F_c map (contoured at 1σ) encompassing the trans-configuration is colored gray. Refinement with the trans-configuration modeled into the 0.5-, 1-, and 2-min structures, reverted to the cis-form in all cases (see “Experimental procedures” for more details). B, the 1-min structure is shown with d-G3P bound in the unproductive cis-form (wheat). The 3(S)-4(R) stereochemistry of the condensation product, FBP, requires prior rotational isomerization about the d-G3P C1–C2 bond, yielding a putative d-G3P trans-configuration (model shown in cyan). The position of trans-d-G3P in the active site was energy-minimized using geometric restraints generated by refmac5 (67). C, superposition of the reported d-G3P configurations shows a hydrogen bond with Glu-189 (dotted cyan line) in the modeled trans-configuration, which, together with Lys-146, orients the carbonyl O1 to promote efficient proton transfer to the polarized d-G3P carbonyl that would otherwise be unproductive in the cis-form.

Figure 4.

l-G3P model reveals basis for enantiomeric discrimination in class I aldolase. A, the l-enantiomer of the bound d-G3P (yellow) was superimposed on the d-G3P geometry from the 1-min structure. Favorable linear syn hydrogen bonds are depicted as green dashes. The steric repulsion resulting from proximity of l-G3P O2 to surrounding oxygens (DHAP O3 oxygen, Asp-34 carboxylate oxygens) is depicted as dark dashes. The trans-configuration (blue) for l-enantiomer modeled using the trans-d-G3P model yields repulsions comparable with those observed for the cis-configuration. B, SBP was modeled into the active site using the FBP Schiff base intermediate as a template. Clashes (dark dashes) and favorable hydrogen bonds (green dashes) are illustrated.