The enzyme
citrate synthase (E.C. 2.3.3.1 [previously 4.1.3.7]) exists in nearly all living cells and stands as a pace-making enzyme in the first step of the
Citric Acid CycleThe citric acid cycle — also known as the tricarboxylic acid cycle , the Krebs cycle, or more rarely, the Szent-Györgyi-Krebs cycle — is a series of enzyme-catalysed chemical reactions, which is of central importance in all living cells that use oxygen as part of cellular respiration...
(or Krebs Cycle). Citrate synthase is localized within eukaryotic cells in the
mitochondrial matrixIn the mitochondrion, the matrix contains soluble enzymes that catalyze the oxidation of pyruvate and other small organic molecules.The mitochondrial matrix also contains the mitochondria's DNA and ribosomes. The word "matrix" stems from the fact that this space is viscous, compared to the...
, but is encoded by nuclear
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
rather than mitochondrial. It is synthesized using cytoplasmic ribosomes, then transported into the mitochondrial matrix.
The enzyme
citrate synthase (E.C. 2.3.3.1 [previously 4.1.3.7]) exists in nearly all living cells and stands as a pace-making enzyme in the first step of the
Citric Acid CycleThe citric acid cycle — also known as the tricarboxylic acid cycle , the Krebs cycle, or more rarely, the Szent-Györgyi-Krebs cycle — is a series of enzyme-catalysed chemical reactions, which is of central importance in all living cells that use oxygen as part of cellular respiration...
(or Krebs Cycle). Citrate synthase is localized within eukaryotic cells in the
mitochondrial matrixIn the mitochondrion, the matrix contains soluble enzymes that catalyze the oxidation of pyruvate and other small organic molecules.The mitochondrial matrix also contains the mitochondria's DNA and ribosomes. The word "matrix" stems from the fact that this space is viscous, compared to the...
, but is encoded by nuclear
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
rather than mitochondrial. It is synthesized using cytoplasmic ribosomes, then transported into the mitochondrial matrix. Citrate synthase is commonly used as a quantitative
enzymeEnzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at...
marker for the presence of intact mitochondria.
Citrate synthase
catalyzesCatalysis is the process in which the rate of a chemical reaction is either increased or decreased by means of a chemical substance known as a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. The catalyst may participate...
the
condensation reactionA condensation reaction is a chemical reaction in which two molecules or moieties combine to form one single molecule, together with the loss of a small molecule. When this small molecule is water, it is known as a dehydration reaction; other possible small molecules lost are hydrogen chloride,...
of the two-carbon
acetateAn acetate, or ethanoate, is either a salt or ester of ethanoic acid.In chemistry, the abbreviation Ac refers to the acetyl group. The anion and the functional group may be written as −OAc and AcO−, or OAc respectively...
residue from acetyl coenzyme A and a molecule of four-carbon oxaloacetate to form the six-carbon
citrateA citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid...
. Oxaloacetate will be regenerated after the completion of one round of the Krebs Cycle.
acetyl-CoAAcetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main use is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. In chemical structure, acetyl-CoA is the thioester between coenzyme A and acetic...
+ oxaloacetate + H2OH2O is the chemical formula for water and is also used as an abbreviation for the word "water". H2O or H2O It may also refer to:* H2O , a punk band...
→ citrateA citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid...
+ CoA-SHCoenzyme A is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle...
Oxaloacetate is the first substrate to bind to the enzyme. This induces the enzyme to change its conformation, and creates a binding site for the
acetyl-CoAAcetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main use is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. In chemical structure, acetyl-CoA is the thioester between coenzyme A and acetic...
. Only when this citroyl-CoA has formed will another conformational change cause thioester
hydrolysisHydrolysis is a chemical reaction during which one or more water molecules are split into hydrogen and hydroxide ions, which may go on to participate in further reactions. It is the type of reaction that is used to break down certain polymers, especially those made by step-growth polymerization...
and release coenzyme A. This ensures that the energy released from the thioester bond cleavage will drive the condensation.
Structure
Citrate synthase's 437 amino acid residues are organized into two main subunits, each consisting of 20 alpha-helices. These alpha helices compose approximately 75% of citrate synthase's
tertiary structureIn biochemistry and chemistry, the tertiary structure of a protein or any other macromolecule is its three-dimensional structure, as defined by the atomic coordinates.-Relationship to primary structure:...
, while the remaining residues mainly compose irregular extensions of the structure, save a single beta-sheet of 13 residues. Between these two subunits, a single cleft exists containing the active site. Two binding sites can be found therein: one reserved for citrate or oxaloacetate and the other for Coenzyme A. The active site contains three key residues: His274, His320, and Asp375 that are highly selective in their interactions with substrates. The image to the right highlights the three key amino acids of citrate synthase's active site in its open state (the substrate is absent). The specific atoms involved in interactions are designated by color, and both a drawing and video of their mechanism can be found in the section labeled "Mechanism" below.
The images to the left display the tertiary structure of citrate synthase in its opened and closed form. The enzyme changes from opened to closed with the addition of one of its substrates (such as oxaloacetate).
Mechanism
Citrate Synthase has three key amino acids in its
active siteThe active site of an enzyme contains the catalytic and binding sites. The structure and chemical properties of the active site allow the recognition and binding of the substrate....
which catalyze the conversion of
acetyl-CoAAcetyl-CoA is an important molecule in metabolism, used in many biochemical reactions. Its main use is to convey the carbon atoms within the acetyl group to the citric acid cycle to be oxidized for energy production. In chemical structure, acetyl-CoA is the thioester between coenzyme A and acetic...
(H
3CO-SCoA) and oxaloacetate (COO-CH
2COCOO-) into
citrateA citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid...
(COO-CH
2COHCOOCH
2COO-) and H-SCoA in an
aldol condensationAn Aldol condensation is an organic reaction in which an enolate ion reacts with a carbonyl compound to form a β-hydroxyaldehyde or β-hydroxyketone, followed by dehydration to give a conjugated enone....
reaction. This conversion begins with the negatively charged oxygen in Asp375’s R-group deprotonating acetyl CoA’s alpha carbon. This pushes the e- to form a double-bond with the
carbonyl carbonIn organic chemistry, a carbonyl group is a functional group composed of a carbon atom double-bonded to an oxygen atom : C=O.The term carbonyl can also refer to carbon monoxide as a ligand in an inorganic or organometallic complex In organic chemistry, a carbonyl group is a functional group...
, which in turn forces the C=O up to pick up a proton for the oxygen from one of the nitrogens in the R-group of His274. This neutralizes the R-group (by forming a
lone pairA lone pair is a electron pair without bonding or sharing with other atoms. They are found in the outermost electron shell of an atom, so lone pairs are a subset of a molecule's valence electrons...
on the nitrogen) and completes the formation of an
enolEnols are alkenes with a hydroxyl group affixed to one of the carbon atoms composing the double bond. Enols and carbonyl compounds are in fact isomers; this is called keto-enol tautomerism:...
intermediate (CH
2COH-SCoA). At this point, His274’s amino lone pair formed in the last step attacks the proton that was added to the oxygen in the last step. The oxygen then reforms the carbonyl bond, which frees half of the C=C to initiate a nucleophilic attack to oxaloacetate’s carbonyl carbon (COO-CH
2COCOO-). This frees half of the carbonyl bond to
deprotonateDeprotonation is a chemistry term that refers to the removal of a proton from a molecule, forming the conjugate base. The relative ability for a molecule to give up a proton is measured by a pKa value. A low pKa value indicates that the compound is acidic and will easily...
one of His320’s amino groups, which neutralizes one of the nitrogens in its R-group. This
nucleophilic additionIn organic chemistry, a nucleophilic addition reaction is an addition reaction where in a chemical compound a π bond is removed by the creation of two new covalent bonds by the addition of a nucleophile....
results in the formation of citroyl-CoA (COOCH
2CHCOOCH
2COHSCoA
2-). At this point, a water molecule is brought in and is deprotonated by His320’s amino group and
HydrolysisHydrolysis is a chemical reaction during which one or more water molecules are split into hydrogen and hydroxide ions, which may go on to participate in further reactions. It is the type of reaction that is used to break down certain polymers, especially those made by step-growth polymerization...
is initiated. One of the oxygen’s lone pairs nucleophilically attacks the
carbonylIn organic chemistry, a carbonyl group is a functional group composed of a carbon atom double-bonded to an oxygen atom : C=O.The term carbonyl can also refer to carbon monoxide as a ligand in an inorganic or organometallic complex In organic chemistry, a carbonyl group is a functional group...
carbon of citroyl-CoA. This forms a tetrahedral intermediate and results in the ejection of –SCoA as the carbonyl reforms. The –SCoA is protonated to form HSCoA. Finally, the hydroxyl added to the carbonyl in the previous step is deprotonated and citrate (-COOCH
2COHCOO-CH
2COO-) is formed.
This
link connects to a video demonstrating citrate synthase's mechanism from Lehninger's Principles of Biochemistry page.
Inhibition
The enzyme is inhibited by high ratios of
ATPAdenosine-5'-triphosphate is a multifunctional nucleotide that plays an important role in cell biology as a coenzyme, that is, the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
:
ADPAdenosine diphosphate, abbreviated ADP, is a nucleotide. It is an ester of pyrophosphoric acid with the nucleoside adenosine. ADP consists of the pyrophosphate group, the pentose sugar ribose, and the nucleobase adenine....
, acetyl-CoA:CoA, and NADH:
NADNAD can refer to:* NAD Electronics, a Canadian audio equipment manufacturer * Namibian dollar, the currency of Namibia* National Advertising Division* National Amusement Devices, manufactured amusement park trains...
, as high concentrations of ATP, acetyl-CoA, and NADH show that the energy supply is high for the cell. It is also inhibited by
succinyl-CoASuccinyl-Coenzyme A, abbreviated as Succinyl-CoA or SucCoA, is a combination of succinic acid and coenzyme A.-Source:It is an important intermediate in the citric acid cycle, where it is synthesized from α-Ketoglutarate by α-ketoglutarate dehydrogenase through decarboxylation...
and
citrateA citrate can refer either to the conjugate base of citric acid, , or to the esters of citric acid...
, examples of product inhibition.
The inhibition of citrate synthase by acetyl-CoA analogues has also been well documented and has been used to prove the existence of a single active site. These experiments have revealed that this single site alternates between two forms, which participate in ligase and hydrolase activity respectively.