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From Proteopedia

Introduction

Citrate synthase catalyses the first step of the citric acid cycle: the condensation of acetyl-CoA and oxaloacetate. The enzyme is encoded in the nucleus and subsequently localized to the mitochondrial matrix where it performs its role in cellular respiration. Interestingly, citrate synthase is often used as a quantitative marker of the content of mitochondria, and therefore level of cellular respiration, as citrate synthase's activity within a tissue is normally constant (when expressed per mitochondrial protein) Kuznetsov.

Structure

is a homodimer of its . The A chain is comprised of 429 residues, and with a molecular weight of 51.7 kDa. Kuznetsov

The shows the 23 alpha helices (blue-gray) comprise the vast majority of each of the protein subunits. In contrast, there is only one small anti-parallel beta sheet (orange) per subunit. Not surprisingly, PBDsum further indicates the numerous helices interact with each other extensively, with 50 helix-helix interactions per chain.

The in the backbone are shown in green. The large number of alpha helices mean that a large portion of the backbone is hydrogen bonded to other molecules of the backbone, as opposed to interacting with side chain molecules, water molecules or the ligand. Citrate synthase lacks disulfide bonds, and so they are not shown here.

The are shown in grey, and the are in pink. The ligand is shown in purple. As custom for many globular proteins, most of the hydrophobic residues are buried within the protein while the hydrophillic ones are more on the surface. This is especially true for the of the protein. (hydrophobic are grey and hydrphillic are pink).

Solvent Interaction

The are shown in blue, the protein in cream and the ligand in a light purple. The A chain can be seen to be more heavily solvated on the side opposite the beta sheet, implying that the side with the beta sheet is where the two monomers join to form the fully functional protein. Indeed, the water interaction (with the same color designations) in the (same colors used) indicates this binding location to be the case and the protein to be fairly equally solvated around the entirety of the protein.

Active Site

The (Acetyl-CoA and oxaloacetate) are shown in pink. Citrate synthase requires two ligands to function, which makes sense as it catalyzes a condensation reaction between two molecules. The first ligand to bind is the smaller oxaloacetate, which induces a conformation change to the active form and allows the larger acetyl-CoA ligand to bind and the reaction to proceed.

The are depicted in ball in stick fashion while the rest of the protein is depicted as a ribbon diagram. Many of the residues surrounding the active site are basic or have the ability to hydrogen bond. These characteristics help to stabilize the multiple negative charges and carbonyl oxygens of the ligands.

The , D375, H274, H320 and S244, PBDsum are shown in black. These catalytic residues are especially important in functioning as acids and basis in the condensation reaction the enzyme performs.

Mechanism of Action

The condensation reaction (Acetyl-CoA + H2O + oxaloacetate = citrate + CoA) has been proposed to involve two concerted general acid-base catalysis steps, meaning the catalytic residues serve as the general acids and bases needed. The mechanism is also seen to proceed through an enol (rather than an enolate) intermediate, and exhibit an inversion of sterochemistry at the nucleophilic carbon atom Karpusas.

The two monomers come together to form a binding cleft for oxaloacetate to bind. After oxaloacetate binds the open conformation of the dimer, the protein undergoes an 18 degree rotation that closes the cleft. The conformation change results in the formation of the acetyl-CoA binding site (Voet).

The reaction itself has three main steps: (Voet)

• 1. The enol of acetyl-CoA is generated (the rate limiting step)
• 2. Citryl-CoA is formed in a concerted acid-base catalysis when the newly formed enolate attacks oxaloacetate. Notably, citrate synthase is able to form a covalent intermediate without the aid of a metal co-factor; a unique feature among enzymes.
• 3. Cirtyl-CoA is hydrolyzed to citrate and CoA. This hydrolysis releases 31.5 kJ/mol of energy, which provides the driving force for the reaction.

References

1. Kuznetsov, A.V., Lassnig, B., Gnaiger, E. (2010). Laboratory Protocol Citrate Synthase Mitochondrial Marker Enzyme. Mitochondrial Physiology Network 08.14: 1-10.1

2. Karpusas M, Branchaud B, Remington SJ. (1990). Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A. Biochemistry. Mar 6;29(9):2213-9. 2

3. PBDsum

4. Voet, D., Voet, J., Pratt, C. (2013). Fundamentals of biochemistry: Life at the molecular level. Wiley. p. 561-563.