Citrate synthase reaction

Entry into the Cycle The Citrate Synthase Reaction... 

FIGURE 20.5 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nncieophiiic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis. 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

At least two enzymes compete for acetyl-CoA - the citrate synthase and 3-ke-tothiolase. The affinities of these enzymes differ for acetyl-CoA (Table l),and at low concentrations of it the citrate synthase reaction tends to dominate, provided that the concentration of 2/H/ is not inhibiting. The fine regulation of the citrate synthases of various poly(3HB) accumulating bacteria has been studied [ 14, 47, 48]. They appear to be controlled by cellular energy status indicators (ATP, NADH, NADPH) and/or intermediates of the TCA cycle. The 3-ketothio-lase has also been investigated [10-14,49, 50]. This enzyme is, above all, inhibited by CoASH [10,14,49]. This important feature will be further considered below. 

A. J. Mulholland, W. G. Richards, Modeling the Citrate Synthase Reaction. QM/MM and Small Model Calculations, in Transition State Modeling for Catalysis, D.G. Truhiar, K. Morokuma (eds), American... 

Substrate availability for certain reactions can be optimized by anaplerotic ( topping-up ) reactions. For example, citrate synthase is a key control point of the TCA cycle. The co-substrates of citrate synthase are acetyl-CoA and oxaloacetate (OAA) and clearly, restriction in the availability of either substrate will decrease the rate of the citrate synthase reaction. Suppose, for example, a situation arises when acetyl-CoA concentration is significantly higher than that of OAA, the concentration of the latter can be topped-up and the concentration of acetyl-CoA simultaneously reduced by diverting some of the pyruvate away from acetyl-CoA synthesis (via pyruvate dehydrogenase) to OAA synthesis (via pyruvate carboxylase) as shown in Figure 3.1. The net effect is to balance the relative concentrations of the two co-substrates and thus to promote citrate synthase activity. 

Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the absence of acetyl-CoA, its positive allosteric modulator. Whenever acetyl-CoA, the fuel for the citric acid cycle, is present in excess, it stimulates the pyruvate carboxylase reaction to produce more oxaloacetate, enabling the cycle to use more acetyl-CoA in the citrate synthase reaction. 

Thermodynamics of Citrate Synthase Reaction in Cells Citrate is formed by the condensation of acetyl-CoA with oxaloacetate, catalyzed by citrate synthase ... 

In rat heart mitochondria at pH 7.0 and 25 °C, the concentrations of reactants and products are oxaloacetate, 1 /am acetyl-CoA, 1 /am citrate, 220 /am and CoA, 65 /am. The standard free-energy change for the citrate synthase reaction is —32.2 kJ/mol. What is the direction of metabolite flow through the citrate synthase reaction in rat heart cells Explain. 

Answer Oxygen consumption is a measure of the activity of the first two stages of cellular respiration glycolysis and the citric acid cycle. Initial nutrients being oxidized are carbohydrates and lipids. Because several intermediates of the citric acid cycle can be siphoned off into biosynthetic pathways, the cycle may slow down for lack of oxaloacetate in the citrate synthase reaction, and acetyl-CoA will accumulate. Addition of oxaloacetate or malate (converted to oxaloacetate by malate dehydrogenase) will stimulate the cycle and allow it to use the accumulated acetyl-CoA. This stimulates respiration. Oxaloacetate is regenerated in the cycle, so addition of oxaloacetate (or malate) stimulates the oxidation of a much larger amount of acetyl-CoA. 

Answer Oxaloacetate depletion would tend to inhibit the citric acid cycle. Oxaloacetate is present at relatively low concentrations in mitochondria, and removing it for gluconeogenesis would tend to shift the equilibrium for the citrate synthase reaction toward oxaloacetate. However, anaplerotic reactions (see Fig. 16-15) counter this effect by replacing oxaloacetate. 

Answer The free-energy change of the citrate synthase reaction in the cell is... 

Thus, the citrate synthase reaction is exergonic and proceeds in the direction of citrate formation. 

There are two Krebs cycle inhibitors that are worth mentioning. Malonate inhibits succinate dehydrogenase because of its very similar structure. Fluoro-acetate inhibits cis-aconitase, which is an Fe-S enzyme. The fluoroacetate replaces acetate as a substrate in the citrate synthase reaction when this combines with cis-aconitase, however, no further reaction becomes possible. 

Table 4.1 Thermodynamic and cation binding parameter values for reactants involved in citrate synthase reaction...

The source of ACCOA for citrate synthase (reaction 2) is the oxidation of carbohydrate, fatty acid, and amino acid molecules. The COAS generated by citrate synthase is used in the oxidation of substrates (such as pyruvate via pyruvate... 

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. 

Citrate synthase is a highly regulated enzyme. What is one of the ways this citrate synthase reaction is driven forward ... 

Although the equilibrium of this reaction favors malate formation, in vivo the reaction proceeds toward the formation of oxaloacetate, since the latter is rapidly removed by the citrate synthase reaction to initiate the next round of the cycle. 

Scheme 4,18 Effect of the different stabilities of fluorothioester enolates (c/s effect) on the stereochemical course of the citrate synthase reaction on fluoroacetyl-CoA [36], In the active site of the enzyme, the enolate is assumed to be bound as the more stable E isomer, with the most electronegative substituents in their relative cis configuration.

In the cytosol, citrate lyase catalyzes the conversion of citrate + CoA + ATP to acetyl CoA + oxaloacetate + ADP + inorganic phosphate. This reaction requires ATP, as the citrate synthase reaction is effectively irreversible. The resulting acetyl CoA in the cytosol can then act as a direct precursor of fatty-acid synthesis via the action of acetyl CoA carboxylase and FAS, as previously described. 

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