1.9-.ANG. Structures of ternary complexes of citrate synthase with D- and L-malate: mechanistic implications

Abstract

The structures of four isomorphous crystals of ternary complexes of chicken heart citrate synthase with D- or L-malate and acetyl coenzyme A or carboxymethyl coenzyme A have been determined by X-ray crystallography and fully refined at 1.9-A resolution. The structures show that both L-malate and D-malate bind in a very similar way in the presence of acetylCoA and that the enzyme conformation is "closed". Hydrogen bond geometry is suggested to account for the difference in binding constants of the two stereoisomers. The structures suggest that steric hindrance can account for the observation that proton exchange of acetyl coenzyme A with solvent is catalyzed by citrate synthase in the presence of L-malate but not D-malate. The ternary complexes with malate reveal the mode of binding of the substrate acetylCoA in the ground state. The carbonyl oxygen of the acetyl group is hydrogen bonded to a water molecule and to histidine 274, allowing unambiguous identification of the orientation of this group. The structures support the hypothesis that carboxymethyl coenzyme A is a transition-state analogue for the enolization step of the reaction (Bayer et al., 1981) and additionally support proposed mechanisms for the condensation reaction (Karpusas et al., 1990; Alter et al., 1990).

Structure and Mechanism of Citrate Synthase

“…If one tries to rotate the conformation of the acetyl group into the orientation adopted by the carboxylate of the transition state analog carboxymethyl-CoA, one finds that severe steric clashes (approximately 2.7 Â) would occur with the carbonyl oxygen of the acetyl group and C-2 and C-3 of D-malate. On the other hand, these become 2.9 and 3.1 Â with L-malate (8). Although these would still be rather close, this is consistent with the fact that the reaction is very slow.…”

“…As previously described, crystal structures reveal the mode of bind ing of the stereoisomers D-and L-malate at 1.9 Â resolution (8). DMalate has the citrate substructure, whereas the naturally occurring form is L-malate.…”

Acetyl-L-carnitine physical-chemical, metabolic, and therapeutic properties: relevance for its mode of action in Alzheimer's disease and geriatric depression

“…Citrate synthase contains 14 lysine residues. 56 When the positive charge on Lys 219 (exposed to aqueous solution on the membrane surface) of the C-terminal domain of cytochrome C oxidase is removed, the reaction rate is increased. 57 ␣Ketoglutarate dihydrogenase contains three lysines in the lipoyl domain and Lys 43 is the lysine that becomes lipoylated, located at the tip of an exposed ␤-turn.…”

Electrostatic complementarity between proteins and ligands. 2. Ligand moieties

“…(4) Citrate synthase ligands: carboxymethylcoenzyme A (ICSC and 2CSC) and acetylcoenzyme A (3CSC and 4CSC) [11].…”