Crystal Structures and Solution Studies of Oxime Adducts of Mitochondrial Aspartate Aminotransferase

Abstract

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X-ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5'-phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single-step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from lo-' M to M depending on the nature of the side-chain group.The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of L-aspartate in the open and closed enzyme conformation were determined at 0.23-nm and 0.25-nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the structures of both oxime complexes and the 2-methylaspartate adduct. The crystal structures indicate that the interaction between the w-carboxylate of the inhibitor and Arg292* of the neighbouring subunit is mainly responsible for the attainment of nearcoplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived inhibitors, even those containing a carboxylate group, do not induce the 'domain closure' in solution. This is probably due to the absence of the a-carboxylate group in the monocarboxylic hydroxylamine-derived inhibitors, emphasizing that both carboxylates of the substrates L -A s~ and L -G~ are essential for stabilizing the closed form of aspartate aminotransferase.

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

“…2D) shows that the amino group of AOA binds covalently to the C4A atom of PLP, as has also been observed for the interaction of AOA with aspartate aminotransferase (38). The ether oxygen atom (O1) is close to Lys-280 (2.9 Å), and the carboxylate group binds via a salt bridge to the N⑀ and N2 atoms of Arg-54.…”

Biochemical Properties and Crystal Structure of a β-Phenylalanine Aminotransferase from Variovorax paradoxus

“…The structure of VpAT with AOA ( Fig. 3A and B) shows that the amino group of the inhibitor covalently binds to the C-4A atom of the PLP cofactor, as also described for aspartate aminotransferase (73) and recently for MesAT (48). The ether-oxygen atom (O1) of AOA is positioned close (3.0 Å) to the ε-amino group of residue K267.…”

The Structure of ll-Diaminopimelate Aminotransferase from Chlamydia trachomatis: Implications for Its Broad Substrate Specificity

“…20,21,23 Because each Arg residue comes from different domains (the LD and the SD), the binding of substrates brings each domain together to form the closed conformation. 24 The interactions between the two Arg residues and the dicarboxylic acids are so important for the domain closure that the lack of one of the carboxylate groups in substrates would hinder formation of the closed conformation. 24 Although it is uncertain whether the substrate binding induces the closing motion of CtDAP-AT, the necessity for the closing motion in CtDAP-AT for catalysis is quite obvious.…”