Mechanism of inactivation of Escherichia coli aspartate aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid .

The chemical balance of two neurotransmitters, L-glutamate and ©–aminobutyric acid (GABA), is critical in the central nervous system (CNS). Disturbance of this balance is thought to be the cause of a number of neurological disorders, such as depression, epilepsy, and schizophrenia, among others. In an effort to fight these diseases, GABA aminotransferase (GABA-AT) has emerged as a potential drug target. A number of compounds targeting GABA-AT are mechanism-based inhibitors that form covalent adducts to the target enzyme. In some cases such inhibitors have demonstrated their therapeutic potential to treat neurological disorders(1, 2). Although most of those compounds are quite potent and can have a long acting effect, they are often toxic or create severe side effects due to lack of specificity for the target enzyme(3). One compound that has proven to be a potent irreversible inhibitor of GABA-AT is the natural product gabaculine(4). However, gabaculine is not specific for GABA-AT and inactivates a number of other enzymes that function with a similar mechanism as GABA-AT(5). Efforts to build specificity into a compound that would target GABA-AT have been partially successful(3, 6). To design new drugs with better efficacy, the detailed inhibition mechanisms of these compounds have been analyzed. Investigations of the inhibition mechanism of gabaculine with PLP-dependent aminotranferases, both biochemically and structurally, showed that the compound can form an irreversible, aromatic adduct with the pyridoxal phosphate cofactor of GABA-AT or ornithine aminotransferase (Figure 1)(7, 8). A second natural product that takes advantage of the same type of aromatization mechanism when inactivating PLP-dependent enzymes is cycloserine(9–11) (Figure 1). Again, a lack of specificity makes it less useful as a drug. FIGURE 1 Aromatic adducts formed between PLP-dependent enzymes and the inactivators gabaculine (with GABA-AT (6), ornithine-AT (8)) and cycloserine (with D-aAT (10), alanine racemase (9), GABA-AT (7)). In order to build higher specificity for GABA-AT into these types of compounds, analogs have been synthesized that are expected to take advantage of the same mechanism-based reactivity as gabaculine or cycloserine. Two of these compounds are (S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid (S-ADTA)(12) and (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (S-ADFA)(13). Both have been investigated for their potential to undergo an aromatization mechanism(7, 14). Besides aromatization, a number of other mechanisms are considered possible for inactivation by these compounds, including the formation of adducts between the inhibitor and an enzymatic group(7, 14) (Figure 2). In order to settle the mechanistic questions, efforts have been made to solve the crystal structures of GABA-AT with gabaculine or either of the above two analogs. However, these efforts have not been successful. FIGURE 2 Proposed mechanism of inactivation of GABA-AT by S-ADFA (11). Consequently, the inactivation by S-ADTA and S-ADFA has been studied with a model enzyme, E. coli L-aspartate aminotransferase (L-AspAT). We chose L-AspAT for several reasons. The aspartate aminotransferase reaction is part of the glutamate/GABA metabolism pathway in the CNS, and E. coli L-AspAT catalyzes the same reaction as its human homologs in the CNS. Hence, L-AspAT is a good enzyme model to test the effect of GABA-AT inhibitors such as S-ADTA and S-ADFA on PLP-dependent enzymes in the glutamate/GABA metabolism pathway. In addition, L-AspAT has been reported to crystallize at relatively neutral pH values(15) whereas GABA-AT only crystallized at low pH(16). Deductions about mechanism could be misleading if the reaction is pH sensitive. As a result, the crystallographic studies on the inhibition of L-AspAT by S-ADFA can be carried out at a pH that is more representative of physiological conditions. The inactivation products for the reaction of S-ADTA with L-AspAT have been identified and shown to take two forms: an irreversible, aromatic adduct of the inhibitor to PLP and one to K246 (Figure 3)(7, 17). Preference of one over the other is pH dependent. This result indicates that more than one type of aromatization mechanism can occur, albeit both would have the same effect on the activity of the enzyme. FIGURE 3 Aromatic adducts formed between S-ADTA and L-AspAT (13). Despite the chemical similarity between S-ADTA and S-ADFA, further mechanistic studies on S-ADFA against GABA-AT surprisingly did not support an “aromatization mechanism,” resulting in a PLP-S-ADFA adduct(14). A potential inhibition mechanism has been proposed for S-ADFA that involves formation of an adduct to an enzymatic group via a Michael addition, resulting in ring opening (Figure 2). But there has not been enough information to reveal the identity of the enzymatic group involved or the structure of such an adduct. Therefore, to establish the mode of S-ADFA inhibition, structural information is needed. Here we report two nearly identical structures of the complexes between S-ADFA and L-AspAT obtained at pH 7.5 and 8. Compared with the two formed with S-ADTA(17), the inhibitor forms only one adduct with the enzyme, namely, with active site lysine 246, thus irreversibly inactivating the L-AspAT transamination reaction. This adduct is the analog of the major adduct in the L-AspAT-S-ADTA complexes at pH 8(17). Although the two compounds differ only in the identity of the heteroatom in the ring, sulfur in the case of S-ADTA and oxygen in the case of S-ADFA, reaction with the aminotransferase leads to a different product distribution, indicating differences in the details of the mechanisms by which they inactivate the enzyme.