CS

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Abstract

The stereochemical nature of the enzymatic condensation reaction is that the acetyl group of acetyl-Coenzyme A is added to the si-face of the keto moiety of oxaloacetate with the methyl group inversion.

The entire reaction can be subdivided into three chemical reactions: (a) enolization of thioester group of acetyl- Coenzyme A by y proton abstraction from the acetyl moiety to form an enolate anion; (b) condensation of the enolate anion with the carboxyl group of oxaloacetate to form a citryl-thioester; (c) citrate thioester is hydrolyzed to form citrate and coenzyme A. The overall reaction has a turnover number of about 6000 mol s^-1 per active site and an apparent equilibrium constant of 8380 liter mol^-1 at pH 7.2. Oxaloacetate binds first, and the binding constant of acetyl-Coenzyme A increases at least 20-fold. If acetyl-Coenzyme A is allowed to bind first, the binding rate of oxaloacetate is reduced. Citrate synthase (CS) is highly specific and only undergoes condensation reactions when its physiological substrates are oxaloacetate and acetyl-Coenzyme A. In the lyase reaction (the reverse of the condensation reaction), only the citrulline moiety of citrulline-Coenzyme A is cleaved. Only citryl- or malyi-Coenzyme A can be hydrolyzed by this enzyme. From the sequences obtained for the enzymes from pig heart and E. coli, it is known that the pig heart enzyme consists of two identical subunits of 437 amino acids with molecular weight of 48,969. Each subunit has an active site that contains a binding site for oxaloacetate and acetyl-CoA, and these active sites appear to be functionally independent of each other.    

Molecular Structure

The structures for three crystal forms (space groups P 4₁2₁2, C2 and P 4₃2₁2) containing the following enzyme-ligand complexes were determined:

1. citrate synthase from pig heart muscle with no ligands,
2. citrate synthase from pig heart muscle with no ligands,
3. pig heart citrate synthase with bound citrate and Coenzyme A,
4. pig heart citrate synthase with bound oxaloacetate and S-acetonyl Coenzyme A,
5. chicken heart muscle citrate synthase with bound citrate and Coenzyme A, and
6. chicken heart citrate synthase with bound (3R, S)-3, 4 dicarboxy-3-hydroxybutyl-Coenzyme A (a citryl-Coenzyme A analog).

The dimer molecules in all crystal forms, except for the crystals containing oxaloacetate and S-acetonyl Coenzyme A, obey an exact twofold symmetry relationship. In oxaloacetate-bound crystals, the dimer is an asymmetric unit and the subunits are related by local diad. The structure of citrate synthase varies widely among the three crystal forms. The molecule has one "open" and two "closed" conformations. The two closed conformations look similar on the surface, but the packing of the interior side chains is somewhat different.

Figure 1. (a) Space-filling representation of the open form dimer of citrate synthase. (b) Space-filling representation of both closed forms of the dimer. The point of view is down the twofold axis (Wiegand, G.; Remington, S.J. 1986)

Citrate/Oxaloacetate Binding Site

The active site exists in the cleft between the two domains of the subunit. Binding sites for citrate or oxaloacetate and coenzyme A are observed in the closed form of the enzyme. In the C2 crystal form, citrate binds deep in the cleft between the large and small domains and is completely inaccessible to the solvent due to the presence of coenzyme A. The citrate molecule is hydrogen-bonded or salt bridged to three histidine residues (His 174, His 238 and His 320) and three arginine residues (Arg 329, Arg 401 and Arg 421 from the other subunit). The active site is predominantly polar, but non-polar interactions may help determine the extreme substrate specificity of citrate synthase and may act as a trigger for conformational changes, for example, Phe 397 forms an unusual edge-on interaction with the citrate molecule. The active site can accommodate oxaloacetate as it does citrate. The axes of helices K and L point towards bound citrates with opposite polarities, so their electric fields may contribute to citrate binding and polarization.

Figure 2. Stereo drawing of the citrate binding site in the monoclinic model (Wiegand, G.; Remington, S.J. 1986)