Transamination specificity

Assay of Transamination. Since the sum of keto acids and amino acids does not change in a transamination, specific reactions are required to assay the reaction products. Some of the methods used are oxidation of a-ketoglutarate to succinate and determination of succinate with succinic dehydrogenase decarboxylation of oxalacetate with aniline citrate decarboxylation with specific amino acid decarboxylases separation of products on paper chromatograms and spectrophotometric determination of those keto acids that exhibit specific absorption. 

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. 

Compartmentation of these reactions to prevent photorespiration involves the interaction of two cell types, mescrphyll cells and bundle sheath cells. The meso-phyll cells take up COg at the leaf surface, where Og is abundant, and use it to carboxylate phosphoenolpyruvate to yield OAA in a reaction catalyzed by PEP carboxylase (Figure 22.30). This four-carbon dicarboxylic acid is then either reduced to malate by an NADPH-specific malate dehydrogenase or transaminated to give aspartate in the mesophyll cells. The 4-C COg carrier (malate or aspartate) then is transported to the bundle sheath cells, where it is decarboxylated to yield COg and a 3-C product. The COg is then fixed into organic carbon by the Calvin cycle localized within the bundle sheath cells, and the 3-C product is returned to the mesophyll cells, where it is reconverted to PEP in preparation to accept another COg (Figure 22.30). Plants that use the C-4 pathway are termed C4 plants, in contrast to those plants with the conventional pathway of COg uptake (C3 plants). 

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 designed construct, as such, may not influence subsequent steps of transamination due to loss of the strong association between the coenzyme and the synthetic peptide after amino acid binding. However, the selectivity of the peptide for pyridoxal phosphate reveals the potential power of peptide design and the importance of secondary binding interactions for defining the formation of specific binary complexes. 

For all these amino acids, the first reaction involves transamination followed by a specific catabolic pathway. 

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. 

In this transamination, the effect of para substitient groups has been studied using fluorinated phenylpyruvic acids and L-aspartic acid. From these results, the migratory preference is H > F > Cl > Br > CF3. This order has been attributed to the bulkiness of the substituted group [57]. Direct amination of p-substituted succinic acid with phenylalanine ammonialyase (EC 4.3.1.5) has suggested very high substrate specificity that the order of reaction rate is m-F o-F P-p-F >CF3. 

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.

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. 

Previously, AAT had been transformed into an L-tyrosine aminotransferase (TAT) by site-specific mutation of up to six amino acid residues lining the active site of wild-type AAT. The hextuple AAT-mutant achieved kinetic data towards the transamination of aromatic substrates such as i-phenylalanine within an order of magnitude of wild-type TAT (Onuffer, 1995). 

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 ... 

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