Krebs cycle isocitrate dehydrogenase

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

M. Lancien, S. Ferrario-Mery, Y. Eoux, E. Bismuth, C. Ma.sclaux, B. Hirel, P. Gadal, and M. Hodges, Simultaneous expression of NAD-dependent isocitrate dehydrogenase and other Krebs cycle genes after nitrate resupply to short-term nitrogen starved tobacco. Plant Physiol. 120 1X1 (1999). 

The situation is simpler for odd numbered fatty acyl derivatives as [3-oxidation proceeds normally until a 5-carbon unit remains, rather than the usual 4-carbon unit. The C5 moiety is cleaved to yield acetyl-CoA (C2) and propionyl-CoA (C3). Propionyl CoA can be converted to succinyl CoA and enter the TCA cycle so the entire molecule is utilized but with a slight reduction in ATP yield as the opportunity to generate two molecules of NADH by isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase is lost because succinyl-CoA occurs after these steps in the Krebs cycle (Figure 7.18). 

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. 

Krebs cycle intermediates (Table 5.12) and/or enzymes (Table 5.13). Nevertheless, certain key enzymes, especially aconitase and isocitrate dehydrogenase, are very low in activity or are undetectable in species such as H. diminuta, whereas only very small amounts of 14C02, a characteristic end-product of the TCA cycle, were liberated in vitro from [14C]glucose by the tetrathyridia of Mesocestoides corti (399) and adults of Cotugnia digonopora (618). The classical TCA cycle is, therefore, unlikely to function to any significant extent in these cestodes. 

Four of the Krebs cycle reactions are considered irreversible citrate synthase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, and succinyl-CoA synthase. In two of these, COz is evolved. In one reaction, succinyl-CoA synthase, a substrate-level phosphorylation takes place, in which a high-energy compound, GTP, is generated. Note that in three of the reactions NADH is... 

After comparing the protein profiles of myocardial mitochondria between a chronic restraint stress group and a control group, 11 protein spots were found to change, of which seven were identified. Five of these proteins, carnitine palmitoyltransferase 2, mitochondrial acyl-CoA thioesterase 1, isocitrate dehydrogenase 3 (NAD ) alpha, fumarate hydratase 1, and pyruvate dehydrogenase beta, were foimd to decrease in abimdance following chronic restraint stress with fimctional roles in the Krebs cycle and lipid metabolism in mitochondria. The other two proteins, creatine kinase and prohibitin, increased after chronic restraint stress (liu et ak, 2004). 

Isocitrate dehydrogenase (E.C. 1.1.1.42, IDH) from Escherichia coli and isopropylmalate dehydrogenase (E.C. 1.1.1.85, IMDH) from Thermus thermophilus are involved in Krebs cycle and leucine biosynthesis respectively. Both enzymes catalyze the sequential reactions... 

Aconitase catalyzes the isomerization of citrate to isodtrate, isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate, and a-ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of a-keto-glutarate to succinyl-CoA. Succinyl-CoA and the remaining intermediates are the 4-carbon intermediates of the Krebs cycle. Succinyl thiokinase catalyzes the release of coenzyme A from succinyl-CoA and the production of GTP. Succinate dehydro-... 

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... 

In addition, both 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase are stimulated by a rise in mitochondrial Ca + concentration this is important in stimulating the rate of the Krebs cycle dining exercise. 

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. 

Figure 29.1 shows that the high ratio of NADH NAD in the mitochondrion favours reduction of oxaloacetate to malate in the malate dehydrogenase reaction. It also restricts oxidation in the a-ketoglutarate dehydrogenase and isocitrate dehydrogenase reactions. The result is that Krebs cycle is inhibited. 

Fig. 1-13. Krebs cycle. A condensing enzyme, B Aconitase, C Isocitric dehydrogenase, a-ketogluta-rate decarboxylase, E Succinic dehydrogenase, F F"u-marase, G Malic dehydrogenase...

It is highly probable that the Krebs cycle functions in all cells. Several reports have implied that it does not exist in the skin either because some enzymes essential to its performance could not be found in skin homogenate or because oxygen uptake was not stimulated by the addition of various substrates such as citrate or a-ketoglutarate to the skin slices. However, these first conclusions were followed by contradictory observations a-ketoglutarate and other substrates were later found to stimulate oxygen uptake, and two enzymes of the citric acid cycle—malic isocitrate and succinic dehydrogenase—were detected in rat skin. 

The first breakthrough came when Schneider and Hogeboom [81] directly measured NADP isocitric dehydrogenase activity and demonstrated that only 10% of the total enzyme activity is present in the mitochondrial fraction. According to their estimate, this amount is not sufficient to explain the normal oxidation rate of the Krebs cycle metabolites. Assuming that the NADP isocitric dehydrogenase in the mitochondria is a contaminant, they concluded that at least some of the enzymes involved in the Krebs cycle are extrami-tochondrial. 

A new series of observations seems to reconcile all these points of view. It was confirmed that most of the NADP isocitric dehydrogenase is extramitochon-drial, except for 10% of the enzyme that is truly mitochondrial and not due to contamination by the supernatant NADP isocitric dehydrogenase. But it is apparently the NAD isocitric dehydrogenase which is active in the Krebs cycle, since in mitochondria that have been deprived of their nucleotides, the Krebs cycle activity can be restored by adding NAD but not by adding NADP. NAD isocitric dehydrogenase has been extracted from beef mitochondria. The enzyme is activated by ADP and inhibited by ADP and NADH [83]. 

Various relationships between enzymes of the Krebs cycle and mitochondria are possible. For instance, all enzymes could be enclosed within mitochondrial structures or the enzymes could take part in the structural build-up of the cell. There is no evidence demonstrating that all enzymes of the Krebs cycle are part of the mitochondria. The existence of enzymes with multiple catalytic properties (isocitric dehydrogenase, aconitase, and malic dehydrogenase) and the failure to separate the multiple steps of an overall reaction (pyruvic and a-ketoglutarate oxidation) are sometimes taken as evidence for the participation of the enzyme in the building-up of the mitochondrial structure, but these arguments do not take into account the limitations of the actual biochemical methods, and, therefore, conclusions based upon them are premature. 

In spite of the absence of integral mitochondria, the red cell contains some of the enzymes (fumerase, isocitric dehydrogenase, malic dehydrogenase, and cytochrome oxidase) functioning in the Krebs cycle and electron transport. These enzymes probably represent mitochondrial remnants, and their presence in the mature erythrocyte may be a consequence of their greater stability. Similarly, enzymes concerned with... 

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