Citric acid cycle isocitrate

To measure the activity of an enzyme of the citric acid cycle, isocitrate dehydrogenase, and the effect of enzyme concentration on the rate of reaction. 

C. Citric acid cycle Isocitrate — a-Ketoglutarate cv-Ketoglutarate — Succinyl-Co A 2NADH+H+— 2NADH+H+— 6 ATP 6 ATP ... 

Aconitase catalyzes the citric acid cycle reaction citrate isocitrate... 

In contrast to laboratory reactions, enzyme-catalyzed reactions often give a single enantiomer of a chiral product, even when the substrate is achiral. One step in the citric acid cycle of food metabolism, for instance, is the aconitase-catalyzed addition of water to (Z)-aconitate (usually called ris-aconitate) to give isocitrate. 

Water can add reversibly to o ,/3-unsalurated aldehydes and ketones to yield /3-hydroxy aldehydes and ketones, although the position of the equilibrium generally favors unsaturated reactant rather than saturated adduct. A related addition to an c /S-unsaturated carboxylic acid occurs in numerous biological pathways, such as the citric acid cycle of food metabolism where ds-aconitate is converted into isocitrate by conjugate addition of water to a double bond. 

Isocitric acid, an intermediate in the citric acid cycle of food metabolism, has the systematic name (2/ ,3S)-3-carboxy-2-hydroxypentanedioic acid. Draw the structure. 

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. 

Isocitric acid, in citric acid cycle, 6 633 Isocontour surfaces, discrete, 10 340 Isocorrosion diagrams, 23 784 Isocratic chromatography, 3 827 Isocrotonic acid, physical properties,... 

Aconitase, an unstable enzyme,4 is concerned with the reversible conversion of cis-aconitate to either citric acid or isocitric acid. It may be noted that the entire system of tricarboxylic cycle enzymes are present in the mitochondria separated from cells, and, furthermore, it has been found that the mitochondrial enzymes differ from the isolated enzymes in that the former require no addition of D.P.N. (co-enzyme I) or T.P.N. (co-enzyme II) for activity. Peters suggests that the citrate accumulation is caused by the competitive reaction of the fluorocitrate with aconitase required for the conversion of citrate to isocitrate. This interference with the tricarboxylic acid... 

In the presence of adequate O, the rate of oxidative phosphorylation is dependent on the availability of ADR. The concentrations of ADR and ATR are reciprocally related an accumulation of ADR is accompanied by a decrease in ATR and the amount of energy available to the celL Therefore, ADR accumulation signals the need for ATR synthesis. ADR aUosterically activates isocitrate dehydrogenase, thereby increasing the rate of the citric acid cycle and the production of NADH and FADH. The elevated levels of these reduced coenzymes, in turn, increase the rate of electron transport and ATR synthesis. 

Aconitase [citrate(isocitrate) hydro-lyase, EC 4.2.1.3] is the second enzyme of the citric acid cycle, which plays a central role in metabolism for all aerobic organisms. This enzyme catalyzes a dehydration-rehydration reaction interconverting citrate and 2R,3S-isocitrate via the allylic intermediate ds-aconitate. 

The enzyme isocitrate dehydrogenase is one of the enzymes of the Krebs or citric acid cycle, a major feature in carbohydrate metabolism (see Section 15.3). This enzyme has two functions, the major one being the dehydrogenation (oxidation) of the secondary alcohol group in isocitric acid to a ketone, forming oxalosuccinic acid. This requires the cofactor NAD+ (see Section 11.2). For convenience, we are showing non-ionized acids here, e.g. isocitric acid, rather than anions, e.g. isocitrate. 

Fluoroacetate produces its toxic action by inhibiting the citric acid cycle. The fluorine-substituted acetate is metabolized to fluoroci-trate that inhibits the conversion of citrate to isocitrate. There is an accumulation of large quantities of citrate in the tissue, and the cycle is blocked. The heart and central nervous system are the most critical tissues involved in poisoning by a general inhibition of oxidative energy metabolism. ... 

Phosphorylation of an enzyme can affect catalysis in another way by altering substrate-binding affinity. For example, when isocitrate dehydrogenase (an enzyme of the citric acid cycle Chapter 16) is phospho-rylated, electrostatic repulsion by the phosphoryl group inhibits the binding of citrate (a tricarboxylic acid) at the active site. 

There are two different forms of isocitrate dehydrogenase in all cells, one requiring NAD+ as electron acceptor and the other requiring NADP+. The overall reactions are otherwise identical. In eukaryotic cells, the NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle. The main function of the NADP-dependent enzyme, found in both the... 

We have now covered one complete turn of the citric acid cycle (Fig. 16-13). A two-carbon acetyl group entered the cycle by combining with oxaloacetate. Two carbon atoms emerged from the cycle as C02 from the oxidation of isocitrate and a-ketoglutarate. The energy released by these oxidations was conserved in the reduction of three NAD+ and one FAD and the produc-... 

The overall rate of the citric acid cycle is controlled by the rate of conversion of pyruvate to acetyl-CoA and by the flux through citrate synthase, isocitrate dehydrogenase, and a-lcetoglutarate dehydrogenase. These fluxes are largely determined by the concentrations of substrates and products the end products ATP and NADH are inhibitory, and the substrates NAD+ and ADP are stimulatory. 

Some bacteria, including E. coli, have the full complement of enzymes for the glyoxylate and citric acid cycles in the cytosol and can therefore grow on acetate as their sole source of carbon and energy. The phosphoprotein phosphatase that activates isocitrate dehydrogenase is stimulated by intermediates of the citric acid cycle and glycolysis and by indicators of reduced cellular energy supply (Fig. 16-23). The same metabolites inhibit the protein kinase activity of the bifunctional polypeptide. Thus, the accumulation of intermediates of... 

The same intermediates of glycolysis and the citric acid cycle that activate isocitrate dehydrogenase are allosteric inhibitors of isocitrate lyase. When energy-yielding metabolism is sufficiently fast to keep the concentrations of glycolytic and citric acid cycle intermediates low, isocitrate dehydrogenase is inactivated, the inhibition of isocitrate lyase is relieved, and isocitrate flows into the glyoxylate pathway, to be used in the biosynthesis of carbohydrates, amino acids, and other cellular components. 

The glyoxylate cycle is active in the germinating seeds of some plants and in certain microorganisms that can live on acetate as the sole carbon source. In plants, the pathway takes place in glyoxysomes in seedlings. It involves several citric acid cycle enzymes and two additional enzymes isocitrate lyase and malate synthase. 

The partitioning of isocitrate between the citric acid cycle and the glyoxylate cycle is controlled at the level of isocitrate dehydrogenase, which is regulated by reversible phosphoiylation. 

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. 

Compartmentalization of Citric Acid Cycle Components Isocitrate dehydrogenase is found only in the mitochondrion, but malate dehydrogenase is found in both the cytosol and mitochondrion. What is the role of cytosolic malate dehydrogenase ... 

---