Transaminase equilibrium

Scheme VI. Stereochemical possibilities in the reduction of an L-amino acid-transaminase equilibrium complex with tritiated NaBH4.

The OAA concentration was calculated from the glutamate oxaloacetate transaminase equilibrium. 

Reported equilibriiun constants for purified glutamic-oxalacetic transaminase preparations have been found to range between 3-7.8 ( 9 , 98, 34 , 345). Reported glutamic-pyruvic transaminase equilibrium values are of the order of 1-2.09 ( 9 , 98, 34 , 343, 344, 340). The pH optimum for glutamic-oxalacetic transaminase from animal tissues has been reported as 7.5 ( 9 , 34 ) and approximately 8 ( 94) from plant seedlings, 8.6 (310, 347) from wheat germ, 8 ( 98) from green plants, 6.9 (31 ) from bacteria, 5.8-S.8 (311, 316, 348) from yeast, 7.8 (349) and from N. crassa, 9 (350). 

A new development is the industrial production of L-phenylalanine by converting phenylpyruvic add with pyridoxalphosphate-dependent phenylalanine transaminase (see Figure A8.16). The biotransformation step is complicated by an unfavourable equilibrium and the need for an amino-donor (aspartic add). For a complete conversion of phenylpyruvic add, oxaloacetic add (deamination product of aspartic add) is decarboxylated enzymatically or chemically to pyruvic add. The use of immobilised . coli (covalent attachment and entrapment of whole cells with polyazetidine) is preferred in this process (Figure A8.17). 

Amino acids for infusion solutions are produced by amino group transfer reactions applying transaminases. Here, a major drawback is the equilibrium conversion of only 50%. Therefore,... 

Next to reactions catalyzed by transaminases, hydrolase-catalyzed reactions also lead to limitations regarding the equilibrium. This problem occurs during ester synthesis, because this condensation reaction produces water. The equilibrium is shifted by high amounts of water towards the reactants therefore, an efficient removal is necessary to reach high conversions. Here, two process setups of Unichema Chemie B V will be discussed illustrating in situ product removal [41]. The first setup is based on azeotropic distillation of the water produced... 

Transamination of alanine yields pyruvate catalysed by alanine transaminase (ALT) whilst aspartate produces oxaloacetate catalysed by aspartate transaminase (AST). All transaminase enzymes operate close to a true equilibrium (K eq 1, see Chapter 2) and... 

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... 

Try-70 is conserved in all transaminases1 3 these transaminases utilize Glu a-KG pair as substrates. X-ray studies have shown that Tyr-70 interacts with the phosphate part of the coenzyme. Y70 F mutant was prepared to examine whether or not Tyr-70 could be a catalytic base. The Y70F mutant showed 15% of the activity of the wild-type enzyme thus, it was quite unlikely that Tyr-70 was the catalytic base. However, Tyr-70 was involved in the interaction with the coenzyme, since the affinity towards the coenzyme was significantly reduced in Y70 F mutant.49-513 The equilibrium dissociation constants for the PMP holoenzyme complex were 1.3 nM and 30 nM for the wild-type and mutant... 

Murakami et al. [19] developed an additional vitamin B6 model system with a binding site. They synthesized an octopus cyclophane (27) as a functional model of the protein matrix of transaminase. This cyclophane formed a hydrophobic cavity in water where PLP could be noncovalendy incorporated. Alkylamines having various hydro-phobic chains were employed as substrates, in place of a-amino adds, to evaluate the hydrophobic effect on the Schiff base-forming equilibrium. The Schiff base formation constant was found to depend markedly on the chain length of a substrate in the presence of 27, indicating that the octopus cyclophane can be utilized as an effective ho-loenzyme model capable of forming a ternary complex. 

Answer In these individuals, the usual route for pyruvate metabolism—conversion to acetyl-CoA and entry into the citric acid cycle—is slowed by the decreased capacity for carrying electrons from NADH to oxygen. Accumulation of pyruvate in the tissues shifts the equilibrium for pyruvate-alanine transaminase, resulting in elevated concentrations of alanine in tissues and blood. 

When the amino donor has the L-configuration and an L-amino acid transaminase is used, the product amino acid will have an L-configuration and when the amino donor has the D-configuration and a D-amino transaminase is used, the product amino acid will have a D-configuration. The equilibrium constant for the reaction is typically close to unity. 

In the presence of NAD, L-malic acid is oxidised to oxaloacetate in a reaction catalysed by L-malate deshydrogenase (l-MDH). The reaction equilibrium is forced in the direction of the products by the elimination of oxaloacetate, via its reaction with 1-glutamate, resulting in the production of L-aspartate. This reaction is catalysed by glutamate-oxaloacetate-transaminase (GOT) ... 

L-Amino acid transaminases are ubiquitous in nature and are involved, be it directly or indirectly, in the biosynthesis of most natural amino acids. All three common types of the enzyme, aspartate, aromatic, and branched chain transaminases require pyridoxal 5 -phosphate as cofactor, covalently bound to the enzyme through the formation of a Schiff base with the e-amino group of a lysine side chain. The reaction mechanism is well understood, with the enzyme shuttling between pyridoxal and pyridoxamine forms [39]. With broad substrate specificity and no requirement for external cofactor regeneration, transaminases have appropriate characteristics to function as commercial biocatalysts. The overall transformation is comprised of the transfer of an amino group from a donor, usually aspartic or glutamic acids, to an a-keto acid (Scheme 15). In most cases, the equilibrium constant is approximately 1. 

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 evolution of a transaminase from Arthrohacter citreus to a thermostable transaminase with increased specific activity and decreased inhibition by the amine product was accomplished using error prone polymerase chain reaction (PCR) [64] The reaction of substituted tetralone 75 and isopropylamine to produce substituted (S) aminotetralin 76 was carried out at greater than 50 °C to facilitate the removal of the acetone by product and drive reaction equilibrium (Figure 14.43). 

Outline the problems associated with transaminase catalyzed amination of ketones to produce chiral amines, particularly with respect to product inhibition and equilibrium. Suggest two different methods that can be used to overcome these problems. 

Three key problems are (i) inhibition of the transaminase enzyme by the product amine, (ii) low conversion of the ketone to the amine at equilibrium, and (iii) inhibition ofthe transaminase by the by product from the amine donor (e.g., pyruvate if alanine is used as the amine donor). One way to overcome these problems is to use pyruvate decarboxylase to remove the pyruvate, which is converted to acetaldehyde. 

Enantiospecific reduction of C=N bonds is of interest for the synthesis of a-amino acids and derivatives such as amines. While nonenzymatic reductive amination has been known since 192711, only recently have enzymatic procedures to L-amino acids became established. The reduction can be achieved by different enzymes following different mechanisms, e.g. by pyridoxalphosphate (PLP)-dependent transaminases (E.C. 2.6.1, discussed in Chapter 12.7) or by amino acid dehydrogenases (E.C. 1.4.1) using NADH or NADPH as the cofactor. The synthetic usefulness of the transaminase reaction is diminished by the location of the equilibrium (Keq often is close... 

The equilibrium of these reactions lies almost completely on the side of lactate. However, by trapping the pyruvate in a subsequent reaction catalyzed by the enzyme glutamate-pyruvate transaminase (GPT) in the presence of L-glutamate, the equilibrium can be displaced in favour of pyruvate and NADH (eqn. 2). 

Many plant tissues contain hydroxypyruvate reductase (o-glycerate dehydrogenase) but it is especially active in leaves (Stafford et al., 1954 Stafford and Magaldi, 1954). Tolbert et al. (1970) purified the spinach enzyme and investigated its properties. It catalyzes reduction of hydroxypyruvate to D-glyceric acid by NADH [Eq. (3)]. Although the equilibrium is toward glyceric acid rather than hydroxypyruvate, the presence of sufficient amino donor and an appropriate transaminase would allow serine synthesis by the nonphosphorylated pathway with Eq. (3) as an intermediate step. 

Little is known about the NADH aitd NAD content of the cytosol. In order to estimate the NADH/NAD ratio, the cytosolic contents of malate, aspartate, glutamate and 2-oxoglutarate were determined by nonaqueous fractionation of spinach leaves (Table l). From these values the NADH/NAD ratio was calculated on the reasonable assumption that the reactions catalyzed by the cytosolic malate dehydrogenase and glutamate oxaloacetate transaminase are near to equilibrium. Introducing the equilibrium constants of these enzymes 2.8. 10 at pH 7.0 (4), Kqqt... 

Determination of ALT ALT (formerly glutamate pyruvate transaminase) catalyzes the equilibrium transfer reaction of the amino group from L-alanine to 2-oxoglutarate to form L-glutamate and pyruvate ALT requires pyridoxal phosphate as coenzyme, which acts as an amino carrier. It is found in the main organs, such as the liver, kidney, and heart. The ALT activity in serum is elevated in diseases of the liver. 

The availabihty of a broad variety of oo-TAs together with efficient techniques to shift the equilibrium allows the biocatalytic synthesis of amines from the corresponding ketones via amino-group transfer. The potential of this protocol is demonstrated by a selection of amines, which can be obtained in nonracemic form by using the most prominent oo-transaminases (Scheme 2.227). 

Transaminase Amine syntiiesis High selectivity Equilibrium State of the art... 

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