Citrate condensing enzyme

Citrate (xi)-synthase [EC 4.1.3.7] (also known as citrate condensing enzyme, citrogenase, and oxaloacetate trans-acetase) catalyzes the reaction of acetyl-CoA with oxaloacetate and water to produce citrate and coenzyme A (in which acetyl-CoA <- (pro-35)-CH2COO ). Citrate (re)-synthase [EC 4.1.3.28] catalyzes the same reaction, albeit with the opposite stereochemistry (thus, acetyl-CoA (pro-3R)-CH2COO ). 

In the first reaction of the cycle (17) acetyl-CoA combines with oxaloacetic acid to form citric acid. This reaction is energy-requiring and it is driven at the expense of acetyl-CoA. Reduced coenzyme A, CoA-SH, is released in the process, and the enzyme for the reaction is the citrate condensing enzyme. 

Citrate (si)-synthase, citrate condensing enzyme, citrogenase (EC 4.1.3.7) the tricarboxylic acid cycle enzyme which catalyses the aldol condensation of ox-aloacetate and acetyl-CoA to form citrate. C.s. from E. coli (M, 248,000) consists of 4 subunits (M, 98,000). C.s. from pig or rat heart (M, 98,000) consists of 2 subunits (M, 49,000). 

Cleavage of Tricarboxylic Adds. Both citric and isocitric acids are reversibly split by enzymes found in various bacteria. Citritase splits citrate to oxalacetate and acetate (XXII). CoA does not participate in this reaction. Unlike the reaction catalyzed by the citrate condensing enzyme, which greatly favors citrate synthesis, the standard free enei ... 

Oxaloacetate looking for a partner Thinks active acetate looks OK Condensing enzyme arranging a merger Makes a new citrate, and kicks out CoA. 

The second metabolic pathway which we have chosen to describe is the tricarboxylic acid cycle, often referred to as the Krebs cycle. This represents the biochemical hub of intermediary metabolism, not only in the oxidative catabolism of carbohydrates, lipids, and amino acids in aerobic eukaryotes and prokaryotes, but also as a source of numerous biosynthetic precursors. Pyruvate, formed in the cytosol by glycolysis, is transported into the matrix of the mitochondria where it is converted to acetyl CoA by the multi-enzyme complex, pyruvate dehydrogenase. Acetyl CoA is also produced by the mitochondrial S-oxidation of fatty acids and by the oxidative metabolism of a number of amino acids. The first reaction of the cycle (Figure 5.12) involves the condensation of acetyl Co and oxaloacetate to form citrate (1), a Claisen ester condensation. Citrate is then converted to the more easily oxidised secondary alcohol, isocitrate (2), by the iron-sulfur centre of the enzyme aconitase (described in Chapter 13). This reaction involves successive dehydration of citrate, producing enzyme-bound cis-aconitate, followed by rehydration, to give isocitrate. In this reaction, the enzyme distinguishes between the two external carboxyl groups... 

Scheme 10 General reaction mechanism of the CoA-dependent Claisen-type condensing enzymes, malate synthase, a-isopropylmalate synthase, citrate synthase, and homocitrate synthase.

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. 

It is well known that in the oxidation of pyruvate an active 2-carbon intermediate, known as active acetate, is formed (Gurin and Crandall, 1948 Bloch, 1948 Lehninger, 1950). A suggestion that pantothenic acid is involved in the conversion of acetate to citrate in yeast came from Novelli and Lipmann s (1950) experiments showing a correlation between the ability of the yeast to oxidize acetate or ethanol and the coenzyme A content of the cells. The formation of citrate from acetate and oxaloacetate in cell-free extracts from pigeon liver or yeast was also shown to require coenzyme A (Novelli and Lipmann, 1950 Stern and Ochoa, 1949, 1951). Further study of these enzyme systems led to the isolation of a crystalline condensing enzyme from pig heart (Ochoa et al., 1951) and to a formulation of the mechanism of the enzymatic synthesis of citric acid from... 

Confirmatory evidence for this structure has come from experiments in which citric acid was synthesized from acetyl-CoA and oxaloacetic acid in the presence of the condensing enzyme. Sulfhydryl groups equivalent to the citrate synthesized were released (Stern, 1951). 

This reaction is essentially irreversible and the thioester bond of acetyl-S-CoA (CHgCO-SCoA) has a high free energy of hydrolysis (AG° = —31 kJ —7 5 kcal). This energy is utilized in a condensation reaction of acetyl CoA with the enol form of oxaloacetic acid to produce citric acid and CoASH is liberated. The enzyme mediating this reaction, citrate synthase (condensing enzyme), is the first enzyme of the tricarboxylic acid cycle (Krebs cycle) (Fig. 17.4). 

Long chain acyl-CoA esters cause a competitive inhibition of acetyl-CoA-carboxylase. The elevated levels of long chain fatty acid Co-A esters in diabetes and on starvation may be the basis of the keto-acidosis (Bobtz et al. 1963). Citrate synthase (condensing enzyme) is also inhibited by long chaiii acyl-CoA esters (Wieland et al. 1963 a, b). It appears therefore that the product of fatty acid synthesis is able to regulate the synthesis by means of a feed back mechanism. 

Acetyl phosphate is formed here due to the presence of phosphotransacetyl-ase in these extracts. In the absence of inorganic phosphate the reaction is greatly diminished and no acetyl phosphate is formed. However, if in the absence of phosphate the system is coupled with crystalline-condensing enzyme and oxaloacetate, a rapid synthesis of citrate is observed. This indicates that acetyl-CoA is formed as an intermediate in the absence of acetyl acceptors, such as phosphate or oxaloacetate, the reaction is limited by the CoA, since free CoA cannot be regenerated. In the presence of appropriate acetyl acceptors, free CoA is regenerated and allowed to act catalytically. 

The search for the C, compound formed by condensation of pyruvic and oxaloacetic acids, originally postulated by Krebs, had been fruitless for more than 10 years. In 1949 Ochoa with the collaboration of Joseph Stern isolated an enzyme from heart muscle capable of synthesizing stoichiometric amounts of citric acid from ATP, acetate, CoA and oxaloacetate. First named condensing enzyme , it is now known as citrate synthetase. It was obtained by the Ochoa group in crystalline form, the first enzyme of the cycle to be crystallized. With the pure enzyme in hand, it was clear not only that the product was not a C, compound, but that other alternatives to citrate, namely, m-aconitate and isocitrate, could also be rejected. The citrate synthetase condensation takes place between the methyl group of acetyl-CoA and the keto group of oxaloacetic acid the reaction is reversible. 

Condensation of acetyl-CoA with oxaloacetate to produce citrate is the first reaction of the TCA cycle. This reaction is catalysed by the condensing enzyme (or citrate synthetase) which has been obtained in crystalline finm ... 

Condensing enzyme —condensation of acetyl CoA and oxalace-tate to form citrate. 

Active acetate is linked to a 4-C body, oxalacetate, by the condensing enzyme in a kind of aldol condensation. The product is citrate, which exists in equilibrium with c/s-aconitate and isocitrate, the equilibrium being controlled by aconitase. 

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. 

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

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