The initial reaction between acetyl-CoA and oxaloacetate (C4) to form citrate (C6) is catalyzed by citrate synthase, which forms a carbon–carbon bond between the methyl car bon of acetyl-CoA and the carbonyl carbon of oxaloacetate (Figure 1). The thioester bond of the resultant citryl-CoA is hydrolyzed, releasing citrate and CoASH—an exothermic reaction. The coenzyme A released can be recycled in the conversion of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex.

Fig1. The citric acid (Krebs) cycle. Oxidation of NADH and FADH2 in the respiratory chain leads to the formation of ATP via oxidative phosphorylation. In order to follow the passage of acetyl-CoA through the cycle, the two carbon atoms of the acetyl moiety are shown labeled on the carboxyl carbon (*) and on the methyl carbon (·). Although two carbon atoms are lost as CO2 in one turn of the cycle, these atoms are not derived from the acetyl-CoA that has immediately entered the cycle, but from that portion of the citrate molecule that was derived from oxaloacetate. However, on completion of a single turn of the cycle, the oxaloacetate that is regenerated is now labeled, which leads to labeled CO2 being evolved during the second turn of the cycle. Because succinate is a symmetrical compound, “randomization” of label occurs at this step so that all four carbon atoms of oxaloacetate appear to be labeled after one turn of the cycle. During gluconeogenesis, some of the label in oxaloacetate is incorporated into glucose and glycogen . The sites of inhibition (⊝) by fluoroacetate, malonate, and arsenite are indicated.

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase); the reaction occurs in two steps: dehydration to cis-aconitate and rehydration to isocitrate. Although citrate is a symmetrical 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 the result of channeling—transfer of the product of citrate synthase directly onto the active site of aconitase, without entering free solution. As fatty acid synthesis uses an anaplerotic pathway (pyruvate carboxylase) to add oxaloacetate to the cycle, this in turn will make extra citrate and thus isocitrate. The excess isocitrate will act as a break on aconitase. Because of channeling, citrate is only available in free solution to be transported from the mitochondria to the cytosol for fatty acid synthesis when aconitase is inhibited by accumulation of its product, isocitrate. The “free” citrate can then be exported out of the cycle (cataplerosis) to be a source of acetyl-CoA for fatty acid synthesis. This allows citric acid cycle activity to be maintained to generate ATP and reducing equivalents. This energy will be needed to support the energy demanding process of fatty acid synthesis while providing citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis.

The poison fluoroacetate is found in some of the plants, and their consumption can be fatal to grazing animals. Some fluorinated compounds used as anticancer agents and industrial chemicals (including pesticides) are metabolized to fluoroacetate. It is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate.

Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially, oxalosuccinate, which remains enzyme bound and undergoes decarboxylation to α-ketoglutarate. The decarboxylation requires Mg2+ or Mn2+ ions. There are three isoenzymes of isocitrate dehydrogenase. One, which uses nicotinamide adenine dinucleotide (NAD+), is found only in mitochondria. The other two use NADP+ and are found in mitochondria and the cytosol. Respiratory chain linked oxidation of isocitrate occurs through the NAD+ dependent enzyme.

α-Ketoglutarate undergoes oxidative decarboxylation in a reaction catalyzed by a multienzyme complex similar to that involved in the oxidative decarboxylation of pyruvate (see Figure 17–5). The α-ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex—thiamin diphosphate, lipoate, NAD+, flavin adenine dinucleotide (FAD), and CoA—and results in the formation of succinyl-CoA. The equilibrium of this reaction is so much in favor of succinyl-CoA formation that it must be considered to be physiologically unidirectional. As in the case of pyruvate oxidation (see Chapter 17), arsenite inhibits the reaction, causing the substrate, α-ketoglutarate, to accumulate. High concentrations of ammonia seen in liver disease inhibits α-ketoglutarate dehydrogenase.

Succinyl-CoA is converted to succinate by the enzyme succinate thiokinase (succinyl-CoA synthetase). This is the only example of substrate-level phosphorylation (transfer of a phosphate group bound to the enzyme to GDP or ADP with generation of ATP or GTP) in the citric acid cycle. Tissues in which gluconeogenesis occurs (the liver and kidney) contain two isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP. The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis, and provides a regulatory link between citric acid cycle activity and the withdrawal (cataplerosis) of oxaloacetate for gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that phosphorylates ADP.

When ketone bodies are being metabolized in extrahepatic tissues, there is an alternative reaction catalyzed by succinyl CoA–acetoacetate-CoA transferase (thiophorase), involving transfer of CoA from succinyl-CoA to acetoacetate, forming acetoacetyl-CoA and succinate .

The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the β-oxidation of fatty acids: dehydrogenation to form a carbon–carbon double bond, addition of water to form a hydroxyl group, and a further dehydrogenation to yield the oxo-group of oxaloacetate.

The first dehydrogenation reaction, forming fumarate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial membrane. The enzyme contains FAD and iron-sulfur (Fe-S) protein, and directly reduces ubiquinone in the electron transport chain. Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate. Malate is oxidized to oxaloacetate by malate dehydrogenase, linked to the reduction of NAD+ to form NADH. Although the equilibrium of this reaction strongly favors malate, the net flux is to oxaloacetate because oxaloacetate is rapidly being used. Oxaloacetate is used for multiple reactions (form citrate, leave the mitochondria to be a substrate for gluconeogenesis, or to undergo transamination to form aspartate). NADH is reoxidized to NAD by the respiratory chain.

مواضيع ذات صلة

The Citric Acid Cycle Takes Part in Gluconeogenesis, Transamination, & Deamination

The Citric Acid Cycle Provides Substrates for The Respiratory Chain

Carbohydrates Occur in Cell Membranes & in Lipoproteins

Dyslipidemia and Cardiovascular Risk

Lipoprotein and Lipid Metabolism

Biomedically, Glucose is the Most Important Monosaccharide

Saccharides are Aldehyde or Ketone Derivatives of Polyhydric Alcohols

A Supply of Oxidizable Fuel is Provided in Both The Fed & Fasting States

Many Metabolic Fuels are Interconvertible

The Flux Through Metabolic Pathways Must be Regulated In A Concerted Manner

Metabolic Pathways at The Organ & Cellular Level

Pathways To Process The Major Products Of our Diet