Aspartate oxaloacetate formation

Aspartate aminotransferase 2.6.1.1 L-Aspartate Oxaloacetate Formation of a hydrazone... 

Figure 28-3. Formation of alanine by transamination of pyruvate. The amino donor may be glutamate or aspartate. The other product thus is a-ketoglutarate or oxaloacetate.

It was observed that glutamate and aspartate are diverted predominantly to the synthesis of cell substance rather than to the formation of oxalate. It is not inconsistent to see oc-ketoglutarate being formed from glutamate, while no oxaloacetic acid can be detected in the medium containing aspartate, as the oxaloacetic acid is known to be extremely unstable (2), (62), (Hi). The relatively low yields of oxalic acid, derived... 

The reaction mixture for a coupled assay includes the substrates for the initial or test enzyme and also the additional enzymes and reagents necessary to convert the product of the first reaction into a detectable product of the final reaction. The enzyme aspartate aminotransferase (EC 2.6.1.1), for instance, results in the formation of oxaloacetate, which can be converted to malic acid by the enzyme malate dehydrogenase (EC 1.1.1.37) with the simultaneous conversion of NADH to NAD+, a reaction which can be followed spectropho-tometrically at 340 nm ... 

The fumarate produced in step [4] is converted via malate to oxaloacetate [6, 7], from which aspartate is formed again by transamination [9]. The glutamate required for reaction [9] is derived from the glutamate dehydrogenase reaction [8], which fixes the second NH4 " in an organic bond. Reactions [6] and [7] also occur in the tricarboxylic acid cycle. However, in urea formation they take place in the cytoplasm, where the appropriate isoenzymes are available. 

Although the utility of transaminases has been widely examined, one such limitation is the fact that the equilibrium constant for the reaction is near unity. Therefore, a shift in this equilibrium is necessary for the reaction to be synthetically useful. A number of approaches to shift the equilibrium can be found in the literature.53 124135 Another method to shift the equilibrium is a modification of that previously described. Aspartate, when used as the amino donor, is converted into oxaloacetate (32) (Scheme 19.21). Because 32 is unstable, it decomposes to pyruvate (33) and thus favors product formation. However, because pyruvate is itself an a-keto acid, it must be removed, or it will serve as a substrate and be transaminated into alanine, which could potentially cause downstream processing problems. This is accomplished by including the alsS gene encoding for the enzyme acetolactate synthase (E.C. 4.1.3.18), which condenses two moles of pyruvate to form (S)-aceto-lactate (34). The (S)-acetolactate undergoes decarboxylation either spontaneously or by the enzyme acetolactate decarboxylase (E.C. 4.1.1.5) to the final by-product, UU-acetoin (35), which is meta-bolically inert. This process, for example, can be used for the production of both l- and d-2-aminobutyrate (36 and 37, respectively) (Scheme 19.21).8132 136 137... 

It is clear that biological systems can manage the chemical reactivity of unstable species. For example, oxalo-acetate—a metabolic intermediate in terran metabolism that is a precursor of citric acid, malic acid, and the amino acid aspartic acid—decarboxylates readily, with a half-life measured in minutes at room temperature at neutral pH. The half-life for the decarboxylation of oxaloacetate drops to seconds at high temperatures in pure water. It is not clear how microorganisms that live at high temperatures manage the instability of oxaloacetate, which is a key intermediate in standard biochemistry for the formation of amino acids, such as aspartate, and asparagine. 

Citrate synthase catalyzes the condensation reaction by bringing the substrates into close proximity, orienting them, and polarizing certain bonds. Two histidine residues and an aspartate residue are important players (Figure 1711). One of the histidine residues (His 274) donates a proton to the carbonyl oxygen of acetyl CoA to promote the removal of a methyl proton by Asp 375. Oxaloacetate is activated by the transfer of a proton from His 320 to its carbonyl carbon atom. The concomitant attack of the enol of acetyl CoA on the carbonyl carbon of oxaloacetate results in the formation of a carbon-carbon bond. The newly formed citryl CoA induces additional structural changes in the enzyme. The active site becomes completely enclosed. His 274 participates again as a proton donor to hydrolyze the thioester. Coenzyme A leaves the enzyme, followed by citrate, and the enzyme returns to the initial open conformation. 

The nonessential amino acids are synthesized by quite simple reactions, whereas the pathways for the formation of the essential amino acids are quite complex. For example, the nonessential amino acids alanine and aspartate are synthesized in a single step from pyruvate and oxaloacetate, respectively. In contrast, the pathways for the essential amino acids require from 5 to 16 steps (Figure 24.8). The sole exception to this pattern is arginine, inasmuch as the synthesis of this nonessential amino acid de novo requires 10 steps. Typically, though, it is made in only 3 steps from ornithine as part of the urea cycle. Tyrosine, classified as a nonessential amino acid because it can be synthesized in 1 step from phenylalanine, requires 10 steps to be synthesized from scratch and is essential if phenylalanine is not abundant. We begin with the biosynthesis of nonessential amino acids. 

This removal of the reaction by-product has been achieved through the use of aspartic acid as the amino donor (Scheme 16). The amine group transfer results in the fonnation of oxaloacetate (7), an unstable compound that decarboxylates under the reaction conditions to afford pyruvate (8). As 8 is still an a-keto acid, and is a substrate for a transaminase reaction that results in the production of alanine, another enzyme is used to dimerize the pyruvate. The product of this reaction is acetolactate (9), which, in turn, spontaneously undergoes decarboxylation to result in the overall formation of acetoin (10) as the final by-product. Acetoin is simple to remove and does not participate in any further reactions. Thus, the equilibrium is driven to provide the desired unnatural amino acid that makes the isolation straightforward. 

The increase in glutamate favors transamination of oxaloacetate and limits oxaloacetate availability for phosphoenolpyruvate synthesis. When the [NADH]/[NAD+] ratio is low, malate formation occurs more readily. The cytosolic PEPCK is relatively unaffected by the mitochondrial [NADH]/[NAD+] ratio. Once malate and aspartate are transported to the cytosol and they are reconverted to oxaloacetate, cytosolic PEPCK can convert it to phosphoenolpyruvate. 

Hydrolysis of four high-energy phosphate groups is required for the formation of one molecule of urea. If fumarate is converted to aspartate (by way of malate and oxaloacetate), one NADH molecule is generated that can give rise to three ATP molecules through the electron transport chain, so that the energy expenditure becomes one ATP molecule per each molecule of urea. 

The nonessential amino acids are synthesized by quite simple reactions, whereas the pathways for the formation of the essential amino acids are quite complex. For example, the nonessential amino acids alanine and aspartate are synthesized in a single step from pyruvate and oxaloacetate, respectively. In contrast, the pathways for the essential amino acids require... 

Fig. I. Simplified scheme of the TCA cycle and the formation of glutamate from a-ketoglutarate (a-kg). In astrocytes glutamate is amidated to glutamine in GABAergic neurons some of the glutamate is decarboxylated and enters the GABA shunt. In both neurons and astrocytes anaplerosis occurs via carboxylation of pyruvate to malate or oxaloacetate (ox-ac) from which aspartate is formed.

The same scaffold was used to design catalysts for pyridoxal phosphate-dependent deamination of aspartic acid to form oxaloacetate, one half of the transamination reaction [8], and oxaloacetate decarboxylation [14]. Catalysis was due to binding of pyridoxal phosphate in close proximity to His residues capable of rate limiting 1,3 proton transfer. A two-residue catalytic site containing one Arg and one Lys residue was found to be the most efficient decarboxylation agent, more efficient per residue than the Benner catalyst, most likely due to a combination of efficient imine formation, pK depression and transition state stabilization. 

If the PEP carboxykinase is located in the mitochondrion, the formation of PEP will take place in the mitochondrion and it will be translocated to the cytosol. If the PEP carboxykinase is located in the cytosol, oxaloacetate will be converted to either malate or aspartate and then transported to the cytosol where it will be reconverted to oxaloacetate, which, via the action of PEP carboxykinase, can be converted to PEP. In cases where the enzyme is located both in the mitochondrion and cytosol, as in humans, some of both of these processes take place. To maintain electroneutrality, the transport of these compounds via PEP, aspartate, or malate out of the... 

Commercial preparations of pig heart glutamate-oxaloacetate transaminase have been screened for their ability to transaminate various a-keto acids with l-[ N]glutamate (32). In addition to l-[ N]aspartate, enzyme preparations were able to catalyze the formation of labeled tyrosine, phenylalanine, leucine, and dihydroxyphenylalanine, as demonstrated by HPLC (17). However, these amino acids have not yet been obtained in radiopure form by this method. The -keto acid analogs of valine and tryptophan were not transaminated by the enzyme preparations. Glutamate-oxaloacetate transaminases obtained from several commercial sources have varying abilities to transaminate the -keto acid... 

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