Malate-oxaloacetate reactions

The reaction will proceed toward oxaloacetate formation in the cell if low product concentration is maintained. Oxidation of NADH by the mitochondrial electron transport system and utilization of oxaloacetate in the formation of citrate shifts the malate-oxaloacetate reaction toward oxaloacetate production. 

A typical intramitochondrial concentration of malate is 0.22 mM. If the [NAD ]/[NADH] ratio in mitochondria is 20 and if the malate dehydrogenase reaction is at equilibrium, calculate the intramitochondrial concentration of oxaloacetate at 25°C. 

FIGURE 20.28 The glyoxylate cycle. The first two steps are identical to TCA cycle reactions. The third step bypasses the C09-evolving steps of the TCA cycle to produce snc-cinate and glyoxylate. The malate synthase reaction forms malate from glyoxylate and another acetyl-CoA. The result is that one torn of the cycle consumes one oxaloacetate and two acetyl-CoA molecnles bnt produces two molecnles of oxaloacetate. The net for this cycle is one oxaloacetate from two acetyl-CoA molecnles. 

The malate, oxaloacetate, and fumarate analogues 3-arsonolactate (86), 3-arsonopyruvate (68), and CE)-3-arsonoacrylate (87,88) have been made Ali and Dixon (88) found that the fumarate and malate analogues were not substrates for fumarate hydratase, but competitive inhibitors, arsonoacrylate, H203As—CH=CH—COOH, with fumarate ( / 1.8) and arsonolactate, H203As—CH2—CHOH—COOH, with malate (KJKm 1.6). Incidentally, although phosphonopyruvate is a poor substrate for malate dehydrogenase (89,90), 3-(hydroxyphosphinoyl)pyru-vate, HO—P(H)(0)—CH2—CO—COOH, is much better (89). It proved impossible to show the reverse reaction with arsonolactate (89, 91). 

However, the urea cycle also causes a net conversion of oxaloacetate to fumarate (via aspartate), and the regeneration of oxaloacetate (Fig. 18-12) produces NADH in the malate dehydrogenase reaction. Each NADH molecule can generate up to 2.5 ATP during mitochondrial... 

Fatty acid biosynthesis (and most biosynthetic reactions) requires NADPH to supply the reducing equivalents. Oxaloacetate is used to generate NADPH for biosynthesis in a two-step sequence. The first step is the malate dehydrogenase reaction found in the TCA cycle. This reaction results in the formation of NAD from NADH (the NADH primarily comes from glycolysis). The malate formed is a substrate for the malic enzyme reaction, which makes pyruvate, CO2, and NADPH. Pyruvate is transported into the mitochondria where pyruvate carboxylase uses ATP energy to regenerate oxaloacetate. 

The overall consumption of one molecule of acetyl-CoA in the citric acid cycle is an exergonic process AG° = —60 kJ mol-1. All but two of the individual reactions are exergonic. Step 2 (citrate— isocitrate) and step 8 (malate —>oxaloacetate) are endergonic (Fig. 12-3). 

Both of these reactions are followed by exergonic reactions. The equilibrium of the reaction malate oxaloacetate (step 8) lies in favor of malate formation, so at equilibrium the concentration of oxaloacetate will be low. The next reaction in the cycle (oxaloacetate + acetyl-CoA — citrate) (step 1) is, however, exergonic, and the oxaloacetate is removed to condense with acetyl-CoA. Similarly, the conversion of citrate to isocitrate is endergonic, and at equilibrium the reaction favors the formation of citrate. The next reaction in the cycle (isocitrate—>2-oxoglutarate) is exergonic, and so the isocitrate is removed thus allowing this reaction to proceed. 

Today the metabolic network of the central metabolism of C. glutamicum involving glycolysis, pentose phosphate pathway (PPP), TCA cycle as well as anaplerotic and gluconeogenetic reactions is well known (Fig. 1). Different enzymes are involved in the interconversion of carbon between TCA cycle (malate/oxaloacetate) and glycolysis (pyruvate/phosphoenolpyruvate). For anaplerotic replenishment of the TCA cycle, C. glutamicum exhibits pyruvate carboxylase [20] and phosphoenol-pyruvate (PEP) carboxylase as carboxylating enzymes. Malic enzyme [21] and PEP carboxykinase [22,23] catalyze decarboxylation reactions from the TCA cycle... 

Reminding that the concentration of oxaloacetate must be kept nearly 0 for the synthesis of citrate, malate-to-oxaloacetate reaction may run perpetually even if the concentration ratio [NADH]/[NAD ] is approximately 1 at anaerobic condition. 

Other TCA cycle reactions (isocitrate —> a-ketoglutarate, a-ketoglutarate—E succinyl CoA, malate—>oxaloacetate total of 3 NADH ... 

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are present primarily in the cytoplasm. Oxaloacetate, the product of the pyruvate carboxylase reaction, must thus be transported to the cytoplasm to complete the pathway. Oxaloacetate is transported from a mitochondrion in the form of malate oxaloacetate is reduced to malate inside the mitochondrion by an NADH-linked malate dehydrogenase. After malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD -linked malate dehydrogenase in the cytoplasm (Figure 16.26). The formation of oxaloacetate from malate also provides NADH for use in subsequent steps in gluconeogenesis. Finally, oxaloacetate is simultaneously decarboxylated and phospho-ry lated by phosphoenolpyruvate carboxy kinase to generate phosphoenol pyruvate. The phosphoryl donor is GTP. The GO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. 

The electrons then are used by a hydrogenase enzyme to produce Ha gas. Desuljovibrio can also convert succinate, fumarate, malate, oxaloacetate, or lactate into pyruvate by reactions which resemble fragments of the Krebs cycle (360). 

In this reaction, malate and NAD diffuse into the active site of malate dehydrogenase. Here NAD accepts two electrons from malate oxaloacetate and NADH then diffuse out of the active site. The reduced NADH must then be returned to its NAD form. For each catalytic cycle, a new NAD molecule is needed if the reaction is to occur thus, stoichiometric quantities of the cosubstrate are needed. The reduced form of this coenzyme (NADH) is converted back to the oxidized form (NAD ) via a number of simultaneously occurring processes in the cell, and the regenerated NAD can then participate in another round of catalysis. 

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. 

Determinations of dehydrogenase activities by the O.t. are now conveniently performed in an automatic recording spectrophotometer, so that the change in absorbance at 340 nm is monitored continuously with time, e.g. the activity of malate dehydrogenase is measured from the rate of increase of absorbance at 340 nm due to the production of NADH in the reaction NAD + Malate -> Oxaloacetate + NADH + H. ... 

Oxaloacetate is first reduced to malate by malate dehydrogenase (reaction (9.5)). 

However, by an extraordinary coincidence, a paper (to which our attention was drawn by Professor Krebs) appeared at just this time in the Journal of the American Chemical Society which brought the possible metabolism of glyoxylate very sharply to our notice. D. T. O. Wong and S. J. Ajl reported that extracts of E. coli could promote the condensation of acetyl coenzyme A and glyoxylate to form malate this reaction was, of course, formally analogous to that whereby citrate was formed from acetyl coenzyme A and oxaloacetate. The authors termed the novel enzyme malate synthetase . The blunderbuss experiment we had done suggested that this enzyme was present also in the Pseudomonas... 

The turnover numbers for the forward and backward reactions, measured under the surplus of the corresponding substrate and coenzyme, were found to be 5.5 10 min" and 2.3 10" min" respectively. This means that one act of the forward reaction (L-malate oxaloacetate) takes 10.9 ms, and that of the backward reaction (oxaloacetate L-malate) takes 2.6 ms. 

Finally, citrate can be exported from the mitochondria and then broken down by ATP-citrate lyase to yield oxaloacetate and acetyl-CoA, a precursor of fatty acids (Figure 20.23). Oxaloacetate produced in this reaction is rapidly reduced to malate, which can then be processed in either of two ways it may be transported into mitochondria, where it is reoxidized to oxaloacetate, or it may be oxidatively decarboxylated to pyruvate by malic enzyme, with subse-... 

The second electron shuttle system, called the malate-aspartate shuttle, is shown in Figure 21.34. Oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is oxidized to NAD ). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD to NADH in the matrix. This mitochondrial NADH readily enters the electron transport chain. The oxaloacetate produced in this reaction cannot cross the inner membrane and must be transaminated to form aspartate, which can be transported across the membrane to the cytosolic side. Transamination in the cytosol recycles aspartate back to oxaloacetate. In contrast to the glycerol phosphate shuttle, the malate-aspartate cycle is reversible, and it operates as shown in Figure 21.34 only if the NADH/NAD ratio in the cytosol is higher than the ratio in the matrix. Because this shuttle produces NADH in the matrix, the full 2.5 ATPs per NADH are recovered. 

FIGURE 23.5 Pyruvate carboxyl compartmentalized reaction. Pyruva verted to oxaloacetate in the mitoci Because oxaloacetate cannot be trai across the mitochondrial membrant reduced to malate, transported to tl and then oxidized back to oxaloace gluconeogenesis can continue. 

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. 

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