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.
Highlights
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
The DHAPenamine condenses with D-G3P to form the C3–C4 bond, generating the ketimine intermediate, which undergoes hydrolysis to release FBP
The ordered Uni Bi reaction mechanism is a consequence of structural changes at the molecular level, whereby DHAP binding stabilizes a conformational change with respect to the free enzyme that narrows the active site cleft [2]
Summary
The cryogenic structures trapped only the highly populated intermediates, consistent with biochemical determination of active-site populations near equilibrium [14] This has precluded the trapping of the elusive ternary enzymatic complex, aldolase1⁄7enamine1⁄7DG3P, consistent with incipient aldol addition. This complex comprises ϳ2% of the total bound intermediates [6, 15] Crystallographic characterization of this low-abundance active-site population would afford insight into the structural constraints imposed by the active-site architecture upon the formation of the ternary complex that ensures 3(S)-4(R) stereospecificity in C–C bond formation in FBP aldolases. We reasoned that crystallization conditions favoring acidic pH would provide an opportunity to stabilize reaction intermediates that would otherwise be of low occupancy in human and rabbit muscle aldolase crystals, which are typically grown at neutral pH. Further analyses of these structures revealed the structural basis by which glycolytic aldolases discriminate against the L-G3P enantiomer to ensure aldehyde stereospecificity
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