Isocitrate, synthesis

Step 3 of Figure 29.12 Oxidation and Decarboxylation (2K,3S)-lsocitrate, a secondary alcohol, is oxidized by NAD+ in step 3 to give the ketone oxalosuccinate, which loses C02 to givea-ketoglutarate. Catalyzed by isocitrate dehydrogenase, the decarboxylation is a typical reaction of a /3-keto acid, just like that in the acetoacetic ester synthesis (Section 22.7). The enzyme requires a divalent cation as cofactor, presumably to polarize the ketone carbonyl group. 

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. 

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. 

In isocitrate, there is a CHOH group that is available for oxidation via the coenzyme NAD+ and the enzyme isocitrate dehydrogenase. NADH will then be reoxidized via oxidative phosphorylation, and lead to ATP synthesis. The oxidation product from isocitrate is oxalosuccinate, a -ketoacid that easily... 

The first step is carboxylation of acetyl CoA to malonyl CoA. This reaction is catalyzed by acetyl-CoA carboxylase [5], which is the key enzyme in fatty acid biosynthesis. Synthesis into fatty acids is carried out by fatty acid synthase [6]. This multifunctional enzyme (see p. 168) starts with one molecule of ace-tyl-CoA and elongates it by adding malonyl groups in seven reaction cycles until palmi-tate is reached. One CO2 molecule is released in each reaction cycle. The fatty acid therefore grows by two carbon units each time. NADPH+H is used as the reducing agent and is derived either from the pentose phosphate pathway (see p. 152) or from isocitrate dehydrogenase and malic enzyme reactions. 

B. The rate-limiting step of the TCA cycle is the synthesis of a-ketoglutarate from citrate, catalyzed by isocitrate dehydrogenase (Figure 7—2). 

Answer Anaplerotic reactions replenish intermediates in the citric acid cycle. Net synthesis of a-ketoglutarate from pyruvate occurs by the sequential actions of (1) pyruvate carboxylase (which makes extra molecules of oxaloacetate), (2) pyruvate dehydrogenase, and the citric acid cycle enzymes (3) citrate synthase, (4) aconitase, and (5) isocitrate dehydrogenase ... 

A total of 14 NADPH molecules are utilized to make each palmitate molecule. It comes from three sources the malic enzyme (see earlier) provides one NADPH molecule for every acetyl-CoA molecule generated from citrate. For palmitate, this accounts for eight NADPH molecules. The rest must be derived largely from the hexose monophosphate shunt (see Chapter 18). A minor source of NADPH is cytosolic isocitrate dehydrogenase (see Chapter 18). The synthesis of one palmitate molecule thus requires an equivalent of 7 + (3)14 = 49 ATP molecules. 

The synthesis of most amino acids demands NADPH so that efficient supply of this cofactor is highly relevant in production processes. In C. glutamicum, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate... 

Acetyl CoA Carboxylase Acetyl CoA carboxylase catalyzes the first and rate-Umiting step of fatty acid synthesis carboxylation of acetyl CoA to malonyl CoA. The mammalian enzyme is activated allostericaUy by citrate and isocitrate, and inhibited by long-chain fatty acyl CoA derivatives. It is also activated in response to insulin and inactivated in response to glucagon. 

It is important to note that animals are unable to effect the net synthesis of glucose from fatty acids. Specifically, acetyl CoA cannot be converted into pyruvate or oxaloacetate in animals. The two carbon atoms of the acetyl group of acetyl CoA enter the citric acid cycle, but two carbon atoms leave the cycle in the decarboxylations catalyzed by isocitrate dehydrogenase and a-ketoglutarate dehydrogenase. Consequently, oxaloacetate is regenerated, but it is not formed de novo when the acetyl unit of acetyl CoA is oxidized by the citric acid cycle. In contrast, plants have two additional enzymes enabling them to convert the carbon atoms of acetyl CoA into oxaloacetate (Section 17.4.). 

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