Citric acid cycle, reactions malate oxidation

Malate can move into and out of mitochondria by active transport processes, and the malate produced in this reaction can be used again in the citric acid cycle. However, malate need not be transported back into mitochondria but can be oxidatively decarboxylated to pyruvate by malic mzyrm, which requires NADP+. 

Steps 7-8 of Figure 29.12 Hydration and Oxidation The final two steps in the citric acid cycle are the conjugate nucleophilic addition of water to fumarate to yield (S)-malate (L-malate) and the oxidation of (S)-malate by NAD+ to give oxaloacetate. The addition is cataiyzed by fumarase and is mechanistically similar to the addition of water to ris-aconitate in step 2. The reaction occurs through an enolate-ion intermediate, which is protonated on the side opposite the OH, leading to a net anti addition. 

The final step is the oxidation of (S)-malate by NAD+ to give oxaloacetate, a reaction catalyzed by malate dehydrogenase. The citric acid cycle has now returned to its starting point, ready to revolve again. The overall result of the cycle is... 

Theoretically, a fall in concentration of oxaloacetate, particularly within the mitochondria, could impair the ability of the citric acid cycle to metabolize acetyl-CoA and divert fatty acid oxidation toward ketogenesis. Such a fall may occur because of an increase in the [NADH]/[NAD+] ratio caused by increased P-oxida-tion affecting the equilibrium between oxaloacetate and malate and decreasing the concentration of oxaloacetate. However, pyruvate carboxylase, which catalyzes the conversion of pyruvate to oxaloacetate, is activated by acetyl-CoA. Consequently, when there are significant amounts of acetyl-CoA, there should be sufficient oxaloacetate to initiate the condensing reaction of the citric acid cycle. 

Oxidation of Malate to Oxaloacetate In the last reaction of the citric acid cycle, NAD-linked L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate ... 

Oxaloacetate is formed in the last step of the citric acid cycle by the NAD+-dependent oxidation of L-malate. Can a net synthesis of oxaloacetate from acetyl-CoA occur using only the enzymes and cofactors of the citric acid cycle, without depleting the intermediates of the cycle Explain. How is oxaloacetate that is lost from the cycle (to biosynthetic reactions) replenished ... 

Figure 10-6 Reactions of the citric acid cycle (Krebs tricarboxylic acid cycle). Asterisks designate positions of isotopic label from entrance of carboxyl-labeled acetate into the cycle. Note that it is not the two carbon atoms from acetyl-CoA that are immediately removed as C02 but two atoms from oxaloacetate. Only after several turns of the cycle are the carbon atoms of the acetyl-CoA completely converted to C02. Nevertheless, the cycle can properly be regarded as a mechanism of oxidation of acetyl groups to C02. Green daggers (+) designate the position of 2H introduced into malate as 2H from the medium. FADS designates covalently bound 8-histidyl-FAD (see Chapter 15).

One of the first persons to study the oxidation of organic compounds by animal tissues was T. Thunberg, who between 1911 and 1920 discovered about 40 organic compounds that could be oxidized by animal tissues. Salts of succinate, fumarate, malate, and citrate were oxidized the fastest. Well aware of Knoop s (3 oxidation theory, Thunberg proposed a cyclic mechanism for oxidation of acetate. Two molecules of this two-carbon compound were supposed to condense (with reduction) to succinate, which was then oxidized as in the citric acid cycle to oxaloacetate. The latter was decarboxylated to pyruvate, which was oxidatively decarboxylated to acetate to complete the cycle. One of the reactions essential for this cycle could not be verified experimentally. It is left to the reader to recognize which one. 

Answer Oxygen consumption is a measure of the activity of the first two stages of cellular respiration glycolysis and the citric acid cycle. Initial nutrients being oxidized are carbohydrates and lipids. Because several intermediates of the citric acid cycle can be siphoned off into biosynthetic pathways, the cycle may slow down for lack of oxaloacetate in the citrate synthase reaction, and acetyl-CoA will accumulate. Addition of oxaloacetate or malate (converted to oxaloacetate by malate dehydrogenase) will stimulate the cycle and allow it to use the accumulated acetyl-CoA. This stimulates respiration. Oxaloacetate is regenerated in the cycle, so addition of oxaloacetate (or malate) stimulates the oxidation of a much larger amount of acetyl-CoA. 

These reactions produce two important intermediate compounds, succinate and malate (which is converted into oxaloacetate). The two decarboxylation steps of the citric acid cycle are bypassed, and so there is no oxidation of acetyl-CoA to C02. Two molecules of acetyl-CoA are used, but all the carbon atoms are retained. 

Many other reactions use NADH as a reducing agent or NAD+ as oxidizing agent. Three molecules of NAD+ are used in the citric acid cycle (see the chart on p. 1393). One of these oxidations is the simple transformation of a secondary alcohol (malate) to a ketone (oxaloacetate). 

STEPS 7-8 Regeneration of oxaloacetate. Catalyzed by the enzyme fumarase. conjugate miclcophilic addition of w ater to fumarate yields t-malate in a reaction simUar to tliat of step 2 in the fatty acid j3>oxidation pathway. Oxida-i m with NAD then, gives oxaloacetate in a step catalyzed by malate dehydrogenase, and the citric acid cycle has return to ita starting point, ready to revolve again. 

Note that the standard free energy for this reaction, unlike that for the other steps in the citric acid cycle, is significantly positive. The oxidation of malate is driven by the utilization of the products—oxaloacetate by citrate synthase and NADH by the electron-transport chain. 

Pyrophosphate is rapidly hydrolyzed, and so the equivalent of four molecules of ATP are consumed in these reactions to synthesize one molecule of urea. The synthesis of fumarate by the urea cycle is important because it links the urea cycle and the citric acid cycle (Figure 23.17). Fumarate is hydrated to malate, which is in turn oxidized to oxaloacetate. Oxaloacetate has several possible fates (1) transamination to aspartate, (2) conversion into glucose by the gluconeogenic pathway, (3) condensation with acetyl CoA to form citrate, or (4) conversion into pyruvate. 

When citrate, a citric acid cycle intermediate, moves from the mitochondrial matrix into the cytoplasm, it is cleaved to form acetyl-CoA and oxaloacetate by citrate lyase. The citrate lyase reaction is driven by ATP hydrolysis. Most of the oxaloacetate is reduced to malate by malate dehydrogenase. Malate may then be oxidized to pyruvate and CO, by malic enzyme. The NADPH produced in this reaction is used in cytoplasmic biosynthetic processes, such as fatty acid synthesis. Pyruvate enters the mitochondria, where it may be converted to oxaloacetate or acetyl-CoA. Malate may also reenter the mitochondria, where it is reoxidized to form oxaloacetate. 

Addition of water to a double bond is a reaction that we find in several biochemical pathways. For instance, the citric acid cycle is a key metabolic pathway for the complete oxidation of the sugar glucose and the release of the majority of the energy used by the body. It is also the source of starting materials for the s)m-thesis of the biological molecules needed for life. The next-to-last reaction in the citric acid cycle is the hydration of a molecule of fumarate to produce a molecule called malate. 

Reaction 8. In the final step of the citric acid cycle, malate dehydrogenase catalyzes the reduction of NAD+ to NADH and the oxidation of malate to oxaloacetate. Because the citric acid cycle "began" with the addition of an acetyl group to oxaloacetate, we have come full circle. 

Cosubstrates are loosely bound coenzymes that are reqnired in stoichiometric amonnts by enzymes. The molecnle nicotinamide adenine dinucleotide (NAD) acts as a cosubstrate in the oxidation-rednction reaction that is catalyzed by malate dehydrogenase, one of the enzymes of the citric acid cycle. 

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