Citrate synthase

• OMIM: 118950
• UniProt: O75390

Citrate synthase is that enzyme ( gene associated CS ), which catalyzes the condensation of acetyl-CoA with oxaloacetate into citrate. This reaction is the first step in the citric acid cycle and therefore the CS is essential in the metabolism of all aerobic organisms. Moreover, the reaction, as well as its inverse is part of the citrate -malate, pyruvate cycle to provide NADPH. CS is localized in the mitochondria, the CS gene of the human is, however, in the cell nucleus.

Structurally, the CS of two large domains, which consist almost entirely of α -helices (so-called alpha- protein). The two domains are connected by an intermediate piece. There are known two types of CS. While the citrate synthase in eukaryotes, Gram-positive bacteria and archaea (Type I ) is present as a dimer, form the subunits in Gram-negative bacteria (type II) is a hexamer (see figure below). In addition, in type II one of the domains is compared to the same extended in type I.

The catalyzed reaction:

Oxalacetate is reacted with acetyl -CoA to citrate and CoA with one molecule of water is consumed. The reaction has an enthalpy DG ° ' of -31.5kJ/mol and is due to the rate-determining for the citric acid cycle.

CS is the one without the presence of a metal ion to form a few enzymes that are capable of C-C bonds.

Reaction mechanism

The individual steps during catalysis, the double take place at the dimer are:

This mechanism was proposed in 1982 by Remington and colleagues due to crystallographic studies.

In the reaction ( Figure 2) initially enters the anion of aspartic acid ASP 375 as a catalyzing base, which, together with the polarizing acting on the carbonyl group of the acetyl-CoA - imidazolyl group of histidine HIS 274 from the activated acetic acid 1 one generated by deprotonation as a reactive intermediate to formulierendes enolate 2, which reacts similarly to a aldol addition with the oxaloacetate stereospecifically the S- citryl CoA 3.

The subsequent hydrolysis of the thioester to the prochiral citrate 4 is highly exergonic ( ΔGo < -30 kJ / mol ), which makes that move, the catalysed by citrate synthase overall reaction irreversible. Citrate product of this reaction competes with oxaloacetate for binding to the citrate synthase, so that a high concentration of citrate - despite the high reaction exergonicity - inhibits reaction. There is a competitive product inhibition.

The reproduced in Figure 3 catalytic center contains instead of the naturally occurring acetyl -CoA, a synthetic analogue ( CMX, Carboxymethyldethia -CoA), which, much resembling the structure of the enol form of acetyl -CoA, good in its place at the citrate synthase binds, but is not suited as a reactant in the sense of the Claisen condensation. Consequently, this acetyl -CoA analogs inhibit the reaction, but it makes the position of the substrates in the substrate -enzyme complex visible and thus provides insight into the mechanism of this enzyme-catalyzed reaction.

The mechanism of the Claisen condensation is valid for this reaction is now considered controversial ( even if it is still to be found in most textbooks ), as the abstraction of a proton ( high pK a ) by an aspartate residue ( lower pKa ) is extremely unlikely. According to recent findings, the enzyme both reactants brings through conformational each other so close that the observed proton neither the methyl group can still be assigned to the aspartate residue and a transition is favored by this. This is reminiscent of the mechanism of Low Barrier Hydrogen Bonds, which can be formed only by spatial proximity of donor and acceptor and from -40 to -80 kJ / mol more stable than "normal" hydrogen bonds, but this model is the real reaction mechanism only rudimentary justice. It must be noted at this point that the mechanism of citrate synthase is still not fully understood despite countless papers and publications in this field.