Transaminating enzymes specificity

Figure 14-5 Some reactions of Schiff bases of pyridoxal phosphate, (a) Formation of the quinonoid intermediate, (b) elimination of a (3 substituent, and (c) transamination. The quinonoid-carbanionic intermediate can react in four ways (1—4) if enzyme specificity and substrate structure allow.

Transamination is effected by enzymes specific to particular amino acids. Transamination between aspartate and a-keto-glutarate illustrated in Fig. 22-6 results in the production of oxaloacetate and glutamate. 

Kupiecki and Coon 89) established that the transaminase which forms /3-aminoisobutyric acid also reversibly transaminates /3-alanine. The enzyme specifically requires a-ketoglutarate. From these results the authors suggest that valine is a precursor of /3-alanine in animal tissues. 

Amino acid oxidase oxidatively deaminates the amino acid at the left (one of many) the enzyme specifically catalyzes this particular reaction as described extensively in Chapt. VIII-7. Another possible reaction, decarboxylation, does not occur. The catalysis of CO2 loss requires a different enzyme, and a third reaction, transamination, requires a third enzyme assisting in the exchange of functional groups (the keto group of oxaloacetic acid with the amino group). Obviously each of the three enzymes possesses a characteristic reaction specificity this is true for all enzymes. 

PEP carboxylase occurs in yeast, bacteria, and higher plants, but not in animals. The enzyme is specifically inhibited by aspartate, which is produced by transamination of oxaloacetate. Thus, organisms utilizing this enzyme control aspartate production by regulation of PEP carboxylase. Malic enzyme is found in the cytosol or mitochondria of many animal and plant ceils and is an NADPIT-dependent enzyme. 

Most amino acids lose their nitrogen atom by a transamination reaction in which the -NH2 group of the amino acid changes places with the keto group of ct-ketoglutarate. The products are a new a-keto acid plus glutamate. The overall process occurs in two parts, is catalyzed by aminotransferase enzymes, and involves participation of the coenzyme pyridoxal phosphate (PLP), a derivative of pyridoxine (vitamin UJ. Different aminotransferases differ in their specificity for amino acids, but the mechanism remains the same. 

It was then possible to confirm the existence of two transaminating systems, the original one utilizing pyruvate as amino acceptor, and a second which used oxaloacetate. Both enzymes were purified and found to be very specific for their substrates. The reactions catalyzed were freely reversible. 

The investigation of the aminotransferase activity of apple ACS carried out by Feng et al reveals that it is able to reductively aminate PLP to PMP by transamination of some L-amino acids to their corresponding a-keto acids. The enzyme has shown substrate specificity with the preference of Ala > Arg > Phe > Asp. The addition of excess pyruvate causes a conversion of the PMP form of the enzyme back to the PLP form. The quite unstable PMP form of ACS can generate apoenzyme, which captures PLP to restore its physiologically active form. 

This enzyme catalyzes the transamination of a wide spectrum of a-amino acids and a-keto (or 2-oxo) acids, demonstrating absolute specificity for their D-isomers. The most likely physiologic role is to provide D-amino acids for peptidoglycan synthesis in bacterial cell wall formation. 

GABA synthesis inhibitors act on the enzymes involved in the decarboxylation and transamination of GABA. Glutamic acid decarboxylase (GAD), the first enzyme in GABA biosynthesis, is inhibited easily by carbonyl reagents such as hydrazines [e.g., hydrazinopropionic acid (4.164) or isonicotinic acid hydrazide (4.165)], which trap pyridoxal, the essential cofactor of the enzyme. A more specific inhibitor is allylglycine (4.166). All of these compounds cause seizures and convulsions because they decrease the concentration of GABA. 

Step c of Eq. 24-34 may occur by ring opening to an enol phosphate which ketonizes to the observed product, but step e is a more complex multistep oxidative process.314a,b The last step is transamination to methionine with a glutamine-specific aminotransferase. Another enzyme from Klebsiella converts the same intermediate anion to methylthiopropionate, formate, and CO (Eq. 24-34, step/).315... 

There is an important biochemical counterpart of the deamination reaction that utilizes pyridoxal phosphate, 7, as the aldehyde. Each step in the sequence is catalyzed by a specific enzyme. The a-amino group of the amino acid combines with 7 and is converted to a keto acid. The resulting pyridoxamine then reacts to form an imine with a different a-keto acid, resulting in formation of a new a-amino acid and regenerating 7. The overall process is shown in Equation 25-6 and is called transamination. It is a key part of the process whereby amino acids are metabolized. 

In transamination, the amino group is transferred, by means of specific enzymes, directly to a keto acid (usually 2-oxoglutarate), which forms a substrate for the formation of the new acid. The most studied systems are ... 

Comparison with similar parameters obtained from reactions with free pyridoxamine indicated that IFABP-PX60 catalyzed transamination some 200 times more efficiently. Analysis of the specific kinetic constants kcat and KM indicated that the observed rate acceleration was due mostly to an increase in substrate binding (50-fold), with a smaller effect on the maximal rate (4-fold). While this is an impressive result, the absolute magnitude of kcat/Ku (0.02 s 1 m 1) makes it clear that this catalyst is still quite primitive compared to natural enzyme systems that occasionally operate with catalytic efficiencies near the diffusion limit. 

The procedure reported in Scheme 13.11 describes deracemization of an amino acid involving oxidation with an L-specific enzyme and transamination with a D-amino transferase using D-aspartate 10, which is generated from L-aspartate 11 by aspartate racemase, as the amino donor. The oxidative enzyme is defined as an L-amino acid deaminase, a flavoprotein from Proteus myxofadens [34]. The transamination reaction is shifted towards the product since the oxalacetate 12 formed decarboxylates spontaneously to give pyruvate and carbon dioxide. 

The ring nitrogen of pyridoxal phosphate exerts a strong electron withdrawing effect on the aldimine, and this leads to weakening of all three bonds about the a-carbon of the substrate. In nonenzymic reactions, all the possible pyridoxal-catalyzed reactions are observed - a-decarboxylation, aminotrans-fer, racemization and side-chain elimination, and replacement reactions. By contrast, enzymes show specificity for the reaction pathway followed which bond is cleaved will depend on the orientation of the Schiff base relative to reactive groups of the catalytic site. As discussed in Section 9.3.1.5, reaction specificity is not complete, and a number of decarboxylases also undergo transamination. 

The ratio of transamination decarboxylation is relatively small - of the order of 1 10,000 for glutamate decarboxylase. Nevertheless, this is sufficient to result in significant loss of active enzyme, andMeister (1990) suggested that this may be a control mechanism rather than simply a lack of reaction specificity. 

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