Aminotransferases substrate specificity

Aspartate aminotransferase Substrate specificity 2.1x106-fold increase in cat. effidency towards valine... 

Xian, M., Alaux, S., Sagot, E. and Gefflaut, T., Chemoenz3fmatic synthesis of glutamic acid analogues substrate specificity and S3mthetic apphcations of branched chain aminotransferase from Escherichia coli. J. Org. Chem., 2007, 72, 7560-7566. 

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. 

Substrate specificity of aminotransferases Each amnolrans ferase is specific for one or, at most, a few amino group donors. Aminotransferases are named after the specific amino gap donor, because the acceptor of the amino group is almost always a-ketoglutarate. The two most important aminotrans ferase reactions are catalyzed by alanine aminotransferase ati aspartate aminotransferase (Figure 19.8). 

Many enzymes exist within a cell as two or more isoenzymes, enzymes that catalyze the same chemical reaction and have similar substrate specificities. They are not isomers but are distinctly different proteins which are usually encoded by different genes.22 23 An example is provided by aspartate aminotransferase (Fig. 2-6) which occurs in eukaryotes as a pair of cytosolic and mitochondrial isoenzymes with different amino acid sequences and different isoelectric points. Although these isoenzymes share less than 50% sequence identity, their internal structures are nearly identical.24-27 The two isoenzymes, which also share structural homology with that of E. coli,28 may have evolved separately in the cytosol and mitochondria, respectively, from an ancient common precursor. Tire differences between them are concentrated on the external surface and may be important to as yet unknown interactions with other protein molecules. 

J. J. Onuffer and J. F. Kirsch, Redesign of the substrate specificity of Escherichia cdi aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis, Protein Sci. 1995, 4, 1750-1757. 

E. Sandmeier, E. Marra, and P. Christen, Active-site Arg->Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase, J. Biol. Chem. 

T. Yano, S. Oue, and H. Kagamiyama, Directed evolution of an aspartate aminotransferase with new substrate specificities, Proc. Natl. Acad. Sci. USA... 

Shin and Kim [39] used the accessible surface area of essential amino acid residues of the amine pyruvate aminotransferase and various amino donors and acceptors to explore the active site structure. Their results suggested a model consisting of two pockets, one large and the other small. The size difference between the binding pockets and the strong repulsion for a carboxylate in the small pocket were key determinants of the substrate specificity and stereoselectivity. 

Shin J., Kim B., Exploring the Active Site of Amine Pyruvate Aminotransferase on the Basis of the Substrate Structure-Reactivity Relationship How the Enzyme Controls Substrate Specificity and Stereoselectivity,/. Org. Chem. 2002, 67, 2848-2853. 

The synthesis of chiral a-amino acids starting from a-keto acids by means of a transamination has been reported by NSC Technologies [26, 27]. In this process, which can be used for the preparation of l- as well as D-amino acids, an amino group is transferred from an inexpensive amino donor, e.g., L-glutamic acid, l-22, or L-aspartic acid, in the presence of a transaminase (= aminotransferase). This reaction requires a cofactor, most commonly pyridoxal phosphate, which is bound to the transaminase. The substrate specificity is broad, allowing the conversion of numerous keto acid substrates under formation of the L-amino acid products with high enantioselectivities [28]. 

Rajaram, V., Ratna Prasuna, P, Savithri, H.S., and Murthy, M.R. (2008) Structure of biosynthetic N-acetylornithine aminotransferase from Salmonella typhimu-rium Studies on substrate specificity and inhibitor binding. Proteins 70, 429-441. 

On the other hand, the pathways to both aromatic amino acids, Phe (1)/Tyr (2), in vascular plants are only beginning to be clarified, and could not readily be deduced from bacterial/fungal sequence/comparisons, for example, in terms of substrate(s). Thus the key to fully understanding the pathways to these two aromatic amino acids in plants was to identify the enzymes, as well as to ultimately establish their substrate specificities/ feedback properties that is, of the actual dehydratases, dehydrogenases, and aromatic aminotransferases involved, including how transcriptional regulation is attained. 

Within each of these sublineages, the type of catalyzed reaction was mostly conserved, whereas the substrates varied. For example, all the enzymes in the ATII subfamily are aminotransferases, with the only apparent exception of dialkylglycine decarboxylase, which, however, catalyzes a decarboxylation-dependent transamination (the enzyme cannot directly transaminate its substrate, which lacks an ct-proton, but proceeds to decarboxylate it and then catalyzes a transamination with the decarboxylation product)." Hence, the evolutionary tree shows that, in general, specialization for reaction type came first, whereas the last and shortest phase of the evolutionary history involved specialization for substrate specificity. 

The construction of enzymes with new substrate specificities is now a realistic goal, and some novel approaches have been presented. For example, removal of an active-site histidine by the His-64 Ala mutation in subtilisin results in an enzyme with markedly reduced activity, but one which can be enhanced 400-fold with substrates containing histidine at the PI site (759). Apparently, the substrate histidine assists catalysis by partially compensating for the role of the lost active-site His-64. In a similar study, mutation of Lys-258 to Ala in aspartate aminotransferase produces an enzyme whose activity can be restored by exogenous amines (140). 

Kynurenine aminotransferase (EC 2.6.1.7) is a PLP-dependent enzyme that converts kynurenine to the corresponding a-ketoacid, employing a-ketoglutarate as an electron acceptor. A rapid cyclisation of the reaction product leads to kynurenic acid. By the same way, HK is converted to xanthurenic acid. In the brain, two forms of kynurenine aminotransferase have been found, somewhat differing as regards substrate specificity, affinity, and inhibition. The mechanism of the enzymic transamination of kynurenine and HK has drawn little attention, even if its irreversible character should be an interesting feature. 

Pong Bi Bi reaction mechanism. As they contain PLP, the mechanism is almost certainly similar to that known for the animal aminotransferases (Fig. 1). Details of this mechanism are discussed by Braunstein (1973) and by Metzler (1977). The apoenzyme moiety determines substrate specificity and confers high catalytic efficiency, as well as suppressing side reactions and eliminating the metal requirement characteristic of nonenzymatic transamination. Initially the amino acid binds to an anchoring site on the enzyme. Condensation then takes place between the amino acid and the enzyme pyridoxal-lysine imine to form an aldimine. Following further rearrangements, a ketimine is produced. Ketimine formation is then followed by a hydrolysis to... 

Because few plant enzymes have been purified to a homogeneous state, final and definitive conclusions cannot yet be drawn concerning their substrate specificities. Many aminotransferase preparations hitherto studied have not shown absolute specificity for a single amino donor compound or amino acceptor compound. Where an incompletely purified enzyme catalyzes a multiplicity of reactions, it is obviously difficult to be certain whether the reactions are all being catalyzed by the same enzyme or by different enzymes present in the preparation. With this reservation, it is nevertheless possible to draw a number of conclusions from currently available data. The outer limits of specificity can fi-equently be ascertained, even though it may sometimes be found subsequently that purer preparations will exhibit more restricted substrate specificities. 

Since the substrate specificity of individual aminotransferases may vary widely, it is not known whether the nitrogen atoms of the aspartate family and branched-chain amino acids are derived from a single, or from multiple precursors (Table I). Utilization of a common amino donor in aminotransferase catalyzed reactions would strengthen the biosynthetic relationship among the pathway products, whereas multiple precursors could tend to balance the synthesis of these amino acids with that of other protein precursors in a type of crosspathway or interfamily regulation. 

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