CRYSTAL-STRUCTURES AND SOLUTION STUDIES OF OXIME ADDUCTS OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE

The interaction of mitochondrial aspartate aminotransferase with hydro xylamine and five derivatives (in which the hydroxyl hydrogen is repla ced by the side chain of naturally occurring amino acids) was investig ated by X-ray diffraction as well as by kinetic and spectral measureme nts with the enzyme in solution. The inhibitors react with pyridoxal 5 '-phosphate in the enzyme active site, both in solution and in the cry stalline state, in a reversible single-step reaction forming spectrall y distinct oxime adducts. Dissociation constants determined in solutio n range from 10(-8) M to 10(-6) M depending on the nature of the side- chain group. The crystal structures of the adducts of mitochondrial as partate aminotransferase with the monocarboxylic analogue of L-asparta te 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 cofac tor pyridoxal phosphate were detected between the structures of both o xime complexes and the 2-methylaspartate adduct. The crystal structure s indicate that the interaction between the omega-carboxylate of the i nhibitor and Arg292 of the neighbouring subunit is mainly responsible for the attainment of near-coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe a imed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine-derived i nhibitors, even those containing a carboxylate group, do not induce th e 'domain closure' in solution. This is probably due to the absence of the alpha-carboxylate group in the monocarboxylic hydroxylamine-deriv ed inhibitors, emphasizing that both carboxylates of the substrates L- Asp and L-Glu are essential for stabilizing the closed form of asparta te aminotransferase.