A-Ketoglutarate oxidation

In the conversion of isocitrate to a-ketoglutarate, oxidation and decarboxylation steps occur. Figure 13.9 shows the oxidation step first. Can you suggest a reason why the oxidation step is first ... 

Thiamine pyrophosphate has two important coenzyme roles, both of which focus mostly on carbohydrate metabolism (Figs. 8.26 and 8.27). The active portion of the coen- rae is the thiazole ring. The first step in the oxidative decarboxylation of a-keto acids requires TPP. The two most common examples are pyruvate and a-ketoglutarate, oxidatively decarboxyatedto acetyl CoA and succinyl CoA, respectively. The same reaction is found in the metabolism of the branched-chain amino acids valine, isoleucine, leucine, and methionine. In all cases, TPP is a coenzyme in a mitochondrial multienzyme complex, consisting of TPP, lipoic acid, coenzyme A, FAD, and NAD. Note the number of vitamins required for the oxidative decarboxylation of a-keto acids thiamine (TPP), pantothenic acid (coenzyme A), riboflavin (FAD),and niacin (NAD). 

DR Sanadi, DM Gibson, P Ayengar, L Quellet. Evidence for a new intermediate in the phosphorylation coupled to a-ketoglutarate oxidation. Biochim Biophys Acta 13 146-148, 1954. 

Fig. 3. Prevention of decline of a-ketoglutarate oxidation in deficient homogenates by a-tocopherol and menadione. Medium consists of 40 iimoles of sodium phosphate buffer (pH 7.4), 300 pinoles ofNaCl, 12pinoles of KCl, 4pmoles ofMgSO - 7 HjO, 60 pmoles of substrate, 3 pmoles of DPN (when a-ketoglutarate is substrate). Final volume is 3 ml. Tocopherol was rehomogenized with the homogenate. The curves are the average of five experiments, the standard error being indicated by the vertical lines (Corwin and Schwarz, 1960b).

Lindblad, B., G. Lindstedt, M. Tofft, and S. Lindstedt The Mechanism of a-Ketoglutarate Oxidation in Coupled Enzymatic Oxygenations. J. Amer. Chem. Soc. 91, 4604 (1968). 

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. 

Reaction of Succinyl CoA, The first enzyme-dissociated product of a-ketoglutarate oxidation is succinyl CoA. Succinyl CoA may be metabolized by three known routes the thioester may be hydrolyzed the CoA may be transferred to other acids and a coupled reaction may result in organic phosphate incorporation into ATP. The last reaction is the only substrate-level phosphorylation of the tricarboxylic acid cycle. 

The hydrolysis of succinyl CoA permits a-ketoglutarate oxidation to proceed to completion in the presence of catalytic quantities of CoA, but it results in a waste of the potentially useful thioester bond. One use for the thioester group is to permit metabolism of certain jS-keto acids. A CoA transferase isolated from pig heart is absolutely specific for succinate and succinyl CoA, but only relatively specific for the 8-keto acid (XIV). The rate of reaction is greatest with acetoacetate, and decreases as the chain length is increased from 8-ketovalerate to 8-ketocaproate. Longer chains are not used. Branched chain /3-keto acids can also serve as substrates, but ,j3-unsaturated acids, /3-hydroxy acids, and dicar-boxylic acids cannot accept CoA in this transfer reaction. An enzyme... 

The base in the nucleotide may be either guanine or hypoxanthine in the reaction with the heart muscle enzyme, while the spinach enzyme is specific for adenine. In crude heart muscle preparations nucleoside diphosphate kinase permits catalytic amounts of GTP or ITP to support a-ketoglutarate oxidation in the presence of ADP, which is converted to ATP. 

It has not been determined whether similar reactions are catalyzed by animal or plant enzymes involved in a-ketoglutarate oxidation, or whether similar intermediates remain bound to the enzyme. The formation of CoA-P has been demonstrated in a thiolkinase reaction with extracts of acetone-dried brain, which also contain a transferase that forms acetyl CoA. ... 

Inhibitors of the Citric Add Cycle. Many compounds have been found to inhibit various steps of the citric acid cycle. Two inhibitors are of particular importance. Arsenite specifically inactivates a-ketoglutarate oxidation. This compound has a similar effect on pyruvate oxidation, and it is now believed that arsenite sensitivity is characteristic of systems containing disulfide groups, as found in lipoic acid. Malonate is a competitive inhibitor of succinate oxidation. Besides its usefulness in studies in which it is desirable to prevent further oxidation, this inhibition was instrumental in developing our current concept of competitive inhibition, since the similarity of structures is striking, and the competitive nature of the reaction is easily demonstrated. The specific inhibition of aconitase by fluorocitrate has already been mentioned. Succinate oxidation is inhibited by naturally occurring C-4 dicarboxylic acids in particular oxalacetate has been found to inhibit succinate oxidation in systems that permit oxalacetate to accumulate. 

For each mole of a-ketoglutarate oxidized to succinate and CO2, 1 mole of a-ketoglutarate is reduced to glutamate, and 1 mole of phosphate is incorporated into ATP. 

Lipoic acid is an acyl group carrier. It is found in pyruvate dehydrogenase zard a-ketoglutarate dehydrogenase, two multienzyme complexes involved in carbohydrate metabolism (Figure 18.34). Lipoie acid functions to couple acyl-group transfer and electron transfer during oxidation and decarboxylation of a-keto adds. 

Another important piece of the puzzle came from the work of Carl Martius and Franz Knoop, who showed that citric acid could be converted to isocitrate and then to a-ketoglutarate. This finding was significant because it was already known that a-ketoglutarate could be enzymatically oxidized to succinate. At this juncture, the pathway from citrate to oxaloacetate seemed to be as shown in Figure 20.3. Whereas the pathway made sense, the catalytic effect of succinate and the other dicarboxylic acids from Szent-Gyorgyi s studies remained a puzzle. 

Until now we have viewed the TCA cycle as a catabolic process because it oxidizes acetate units to COg and converts the liberated energy to ATP and reduced coenzymes. The TCA cycle is, after all, the end point for breakdown of food materials, at least in terms of carbon turnover. However, as shown in Figure 20.22, four-, five-, and six-carbon species produced in the TCA cycle also fuel avariety of biosynthetic processes. a-Ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate are all precursors of important cellular species. (In order to par-... 

The catabolism of amino acids provides pyruvate, acetyl-CoA, oxaloacetate, fumarate, a-ketoglutarate, and succinate, ail of which may be oxidized by the TCA cycle. In this way, proteins may serve as excellent sources of nutrient energy, as seen in Chapter 26. 

The half-reactions and reduction potentials in Table 21.1 can be used to analyze energy changes in redox reactions. The oxidation of NADH to NAD can be coupled with the reduction of a-ketoglutarate to isocitrate ... 

One of the biological pathways by which an amine is converted to a ketone involves two steps (1) oxidation of the amine by N.AD+ to give an imine, and (2) hydrolysis of the imine to give a ketone plus ammonia. Glutamate, for instance, is converted by this process into a-ketoglutarate. Show the structure of the imine intermediate, and propose mechanisms for both steps. 

Step 4 of Figure 29.12 Oxidative Decarboxylation The transformation of cr-ketoglutarate to succinyl CoA in step 4 is a multistep process just like the transformation of pyruvate to acetyl CoA that we saw in Figure 29.11. In both cases, an -keto acid loses C02 and is oxidized to a thioester in a series of steps catalyzed by a multienzynie dehydrogenase complex. As in the conversion of pyruvate to acetyl CoA, the reaction involves an initial nucleophilic addition reaction to a-ketoglutarate by thiamin diphosphate vlide, followed by decarboxylation, reaction with lipoamide, elimination of TPP vlide, and finally a transesterification of the dihydrolipoamide thioester with coenzyme A. 

A complete cycle of iron-oxo generation and substrate oxidation with the consumption of one equivalent 02 and one equivalent a-ketoglutarate is necessary for each of the reactions catalyzed by clavaminate synthase. The different substrates utilized by clavaminate synthase adopt slightly different positions relative to the... 

Thiamin has a central role in energy-yielding metabo-hsm, and especially the metabohsm of carbohydrate (Figure 45-9). Thiamin diphosphate is the coenzyme for three multi-enzyme complexes that catalyze oxidative decarboxylation reactions pymvate dehydrogenase in carbohydrate metabolism a-ketoglutarate dehydro-... 

As mentioned in the introductory part, stereochemical course of the conversion of isocitric acid to a-ketoglutaric acid in TCA cycle is completely enantiose-lective although the reaction does not form an asymmetric carbon in the usual metabolic path. If such type of oxidative decarboxylation can be applied to synthetic compounds, it is expected that an entirely new type of asymmetric biotransformation will be developed. 

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