Aspartate aminotransferase substrate specificity

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

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... 

There are numerous transminases, each specific to a given substrate pair. Some may be primarily mitochondrial others, cytosolic. For example, glutamate-oxaloacetate transminase (GOT), also called aspartate aminotransferase (AST), is primarily a mitochondrial enzyme. AST is extensively used in the diagnosis of heart and liver disorders (see Chapter 5). The AST reaction is represented by Equation (20.7). 

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]. 

C.N. Cronin, B.A. Malcolm, J.F. Kirsch, Reversal of substrate charge specificity by site-directed mutagenesis of aspartate aminotransferase,/. Am. Chem. Soc. 1987, 109, 2222-2223. 

In an attempt to reverse the substrate charge specificity for aspartate aminotransferase, an Arg-292 residue which forms a salt bridge with the carboxylate on the side chain of the substrate aspartate was modified to an Asp (60). Model building studies based on the crystal structure of the wild-type enzyme indicated that the Arg-292+ Asp ion pair could be replaced by an Asp-292 + Arg" pair in the mutant (Fig. 7). The specificity of the Arg-292 -> Asp mutant in terms of... 

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). 

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. 

The properties of plant aminotransferases have recently been reviewed by Wightman and Forest (1978) and by Givan (this volume, Chapter 8). Aminotransferases are known to have multiple specificity, and it is possible that asparagine may be acting as a substrate for the ubiquitous aspartate aminotransferases. 

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