Enzymes citrate synthase

Figure 5 A suggested mechanism for the enolization of acetyl-CoA by the enzyme citrate synthase (CS). The keto, enolate, and enol forms of the substrate are shown.

The first sequence is from the enzyme citrate synthase, residues 260-270, which form a buried helix the second sequence is from the enzyme alcohol dehydrogenase, residues 355-365, which form a partially exposed helix and the third sequence is from troponin-C, residues 87-97, which form a completely exposed helix. Charged residues are colored red, polar residues ate blue, and hydrophobic residues are green. 

The consequent interpretation, accepted by Krebs in his review of the tricarboxylic acid cycle in 1943, was therefore that citric acid could not be an intermediate on the main path of the cycle, and that the product of the condensation between oxaloacetate and acetyl CoA would have to be isocitrate, which is asymmetric. This view prevailed between 1941 and 1948 when Ogston made the important suggestion that the embarrassment of the asymmetric treatment of citrate could be avoided if the acid was metabolized asymmetrically by the relevant enzymes, citrate synthase and aconitase. If the substrate was in contact with its enzyme at three or more positions a chiral center could be introduced. 

Figure 7.13 Physiological pathway for fatty acid oxidation. The pathway starts with the hormone-sensitive lipase in adipose tissue (the flux-generating step) and ends with the formation of acetyl-CoA in the various tissues. Acetyl-CoA is the substrate for the flux-generating enzyme, citrate synthase, for the Krebs cycle (Chapter 9). Heart, kidney and skeletal muscle are the major tissues for fatty acid oxidation but other tissues also oxidise them.

A similar aldol reaction is encountered in the Krebs cycle in the reaction of acetyl-CoA and oxaloacetic acid (see Section 15.3). This yields citric acid, and is catalysed by the enzyme citrate synthase. This intermediate provides the alternative terminology for the Krebs cycle, namely the citric acid cycle. The aldol reaction is easily rationalized, with acetyl-CoA providing an enolate anion nucleophile that adds to the carbonyl of oxaloacetic acid. We shall see later that esters and thioesters can also be converted into enolate anions (see Section 10.7). 

The obvious product of the aldol reaction would be the thioester citryl-CoA. However, the enzyme citrate synthase also carries out hydrolysis of the thioester linkage, so that the product is citric acid hence the terminology. The hydrolysis of the thioester is actually responsible for disturbing the equilibrium and driving the reaction to completion. 

That this is not the case for the enzyme citrate synthase suggests we must look at the enzyme binding site to rationalize the different reaction sequence. It becomes clear that the enzyme binding site positions the substrates so that there are acidic and basic amino acid residues available to produce the enolate anion equivalent of acetyl-CoA (shown here as the enol), but not for the oxaloacetate (Figure 13.8). 

Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes citrate synthase, aconitase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. 

The first step has a AG0 of —0.05 kcal/mole, which is close to zero it does not occur to any great extent unless the concentrations of acetyl-coenzyme A (acetyl-CoA) and oxaloacetate are greater than the concentration of citryl-CoA. The second step, however, has a highly favorable AG0 of — 8.4 kcal/mole. When the two steps are combined, AG0 for the overall reaction is about —8.3 kcal/mole, and the equilibrium constant lies far in the forward direction. These two reactions are catalyzed by the enzyme citrate synthase, by a mechanism that ensures that they always occur together. 

This complete oxidative cycle is found in a number of archaebacteria. Halophiles can fulfil their energy requirements by metabolism of amino acids and other nitrogenous compounds, and therefore it is probable that they possess an oxidative citric acid cycle. Aitken and Brown [45] have reported the presence of the cycle s enzymes in Halobacterium halobium and we have found the key enzymes, citrate synthase and succinate thiokinase, in a range of classical and alkaliphilic halophiles [46], Thus, it is probable that the cycle is generally present in this group of archaebacteria, but exhaustive studies have not been carried out. 

Mulholland and Richards [344-346] have carried out ab initio (MP2/6-31-i-G(d) and RHF/6-31+G(d)) and semiempirical (AMI, PM3 and MNDO) molecular orbital calculations focussing on the enzyme citrate synthase. Their calculations were performed on the first stage of the citrate synthase reaction [344], on the substrate oxaloacetate [345] and on a simple model of the condensation reaction [346]. Their aim was to model the nucleophilic intermediate produced by the rate-limiting step, to examine which form of acetyl-CoA is the likely intermediate and how it is stabilised by the enzyme. They have found that the enolate is the likely nucleophilic intermediate in citrate synthase being stabilised by hydrogen bonds. 

What do the enzymes citrate synthase, isocitrate dehydrogenase, and ketoglutarate dehydrogenase have in common ... 

FIGURE 14.15 (cont d). (c) Deduced stereochemical course of the reaction catalyzed by the enzyme citrate synthase. 

Addition to oxaloacetate. Acetyl CoA enters the citric acid cycle in stepj 1 by nucleophilic addition to the ketone carbonyl group of oxaloacetate to give citryl CoA (Section 26.15). The addition is an aldol reaction of an eno-late ion from acetyl CoA, and is catalyzed by the enzyme citrate synthase, as discussed in Section 26.15. Citryl CoA is then hydrolyzed to citrate. 

TCA cycle substrates oxaloacetate and acetyl-CoA and the product NADH are the critical regulators. The availability of acetyl-CoA is regulated by pyruvate dehydrogenase complex. The TCA cycle enzymes citrate synthase. 

CoA and oxaloacetate. Actually, this is another biological example of an aldol condensation reaction. It is catalyzed by the enzyme citrate synthase. The product that is formed is citrate ... 

Acetyl-CoA + Oxaloacetate + H2O <=> Citrate + CoASH + H Enzyme Citrate Synthase... 

Fluorocitrate is an inhibitor of the citric acid cycle enzyme, aconitase. Fluorocitrate is produced by catalytic combination of fluoroacetyl-CoA (see fluoroacetate) with oxaloacetate by the enzyme citrate synthase. 

Cells readily convert fluoroacetate to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase (reaction diagram). Fluoroacetyl-CoA can combine with oxaloacetate to form fluorocitrate in a reaction catalyzed by the citric acid cycle enzyme, citrate synthase. Fluorocitrate is toxic to cells because it inhibits aconitase. 

Important sites of inhibition are the pyruvate dehydrogenase complex, which converts pyruvate into acetyl-CoA isocitrate dehydrogenase, which converts isocitrate into 2-oxoglutarate and 2-oxoglutarate dehydrogenase. The enzyme citrate synthase, which catalyzes the first reaction of the cycle, is also inhibited by ATP. ... 

The TCA cycle begins with condensation of the activated acetyl group and oxaloac-etate to form the 6-carbon intermediate citrate, a reaction catalyzed by the enzyme citrate synthase (see Fig. 20.3). Because oxaloacetate is regenerated with each turn of the cycle, it is not really considered a substrate of the cycle, or a source of electrons or carbon. 

The reaction is catalyzed by the enzyme citrate synthase, originally called condensing enzyme. A synthase is an enzyme that makes a new covalent bond during the reaction, but it does not require the direct input of ATP. It is an exergonic reaction (AG° = -32.8 moP = -7.8 kcal moP ) because the hydrolysis of a thioester releases energy. Thioesters are considered high-energy compounds. 

Affinity chromatography of coenzyme A-dependent enzymes (citrate synthase and succinic thiokinase)... 

In this paper we present oiu" results from studies in homogenates of frozen skeletal muscle. These homogenates were obtained by using glass/glass homogenizers under conditions were the mitochondrial matrix enzymes are completely released as shown by the release of the marker enzyme citrate synthase. ... 

In addition, it has become increasingly evident that there is significant mitochondrial dysfunction and impairment of the oxidative phosphorylation system [29, 41, 66-69]. This impairment is felt to be secondary to inhibition of the Krebs cycle enzymes citrate synthase, aconitase, and isocitrate dehydrogenase by methylcitrate, inhibition of pyruvate carboxylase by methylmalonic acid, and inhibition of pyruvate dehydrogenase complex. 

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