Aspartate Aminotransferase

Many enzymes employ exogenous molecules known as cofactors to assist in executing their chemistry. Sometimes these cofactors are covalently bound to the enzyme and sometimes not. Many types of cofactors are known, and here we will focus on a well-studied example called pyridoxal phosphate (PLP), which often participates in the metabolism of amino acids. PLP, derived from vitamin B6, is a covalently bound cofactor; it is attached to lysine residues by means of a Schiff base or imine linkage as shown at right.

The substrates for most PLP-requiring processes are α-amino acids, and most of the processes take place at the α-carbon position, although some take place at the β- or γ -carbon. The enzymes which use PLP catalyze a wide range of reactions, including racemizations, decarboxylations, and amine transfers. In general, for all three of these classes of reactions at the α-carbon the substrate displaces the lysine and forms an aldimine intermediate with the PLP.

The now very acidic α-proton of the amino acid is abstracted by a basic amino acid residue (often the displaced lysine), with the pyridine ring of PLP acting as an electron sink. For the racemases, a proton is then delivered to the opposite face from the same or a different basic residue with the net result of inversion of configuration at the α-carbon. Attack of the active site lysine effects product release and regenerates the cofactor.

The structure of one PLP-utilizing transaminase, aspartate aminotransferase, is shown in Fig. 8. This enzyme catalyzes the reversible transamination reaction shown below.

Figure 8 Structure of an aspartate aminotransferase. The protein is a homodimer, with one covalently bound pyridoxal phosphate (shown in black) in each of the two subunits. The expanded view shows the cofactor in greater detail. [Adapted from Rhee, S. et al. (1997). “Refinement and comparisons of the crystal structures of pig cytosolic aspartate aminotransferase and its complex with 2-methylaspartate,” J. Biol. Chem. 272, 17293–17302.]

In the transamination reaction, formation of the aldimine intermediate between aspartate and PLP and its deprotonation proceeds as described above for the racemases. However, reprotonation occurs not at the same carbon as in the racemization mechanism but at a position adjacent to the PLP heterocycle.

Hydrolysis releases the product oxaloacetate and generates a new form of the cofactor called pyridoxamine. The reverse reaction is then carried out on the other substrate, α-ketoglutarate, forming glutamate and regenerating the PLP cofactor.

Reactions at the β-position (for example, in threonine dehydatase) or the γ -position (in methionine-γ -lyase) also proceed by means of formation of an aldimine intermediate with the α-carbon of an α-amino acid. Such a survey of PLP-dependent enzymes illustrates the important point that one cofactor can be used for different kinds of transformations. The reactions described all go through a common aldimine intermediate, with the ultimate course of the reaction being controlled by the appropriate substrate specificity and positioning of amino acid side chains. This flexibility allows nature to expand its chemical repertoire with a relatively small set of cofactors.

There are other organic cofactors such as thiamine pyrophosphate and biotin that participate in carbon– carbon bond formation and cleavage, cofactors that participate in reduction/oxidation, or redox, reactions such as nicotinamide and flavin moieties discussed in some of the earlier examples, and still others that are metal based such as vitamin B₁₂ and porphyrin, which is our next topic.