SEMIEMPIRICAL CALCULATIONS OF THE ELECTRONIC ABSORPTION-SPECTRUM OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE .2. THE 2-METHYLASPARTATE COMPLEX

The electronic transitions of the covalent external aldimine adduct of aspartate aminotransferase with the inhibitor L-2-methylaspartate have been calculated using the sermiempirical, all-valence_electron INDO/S method with configuration interaction. The electronic absorption spectra of several protonation states of the system were evaluated and compared with the observed solution spectrum. The model used for the calculation consists of the vitamin B-6 cofactor with covalently bound 2-methylaspartate and nine residues from the protein which interact directly or indirectly with the cofactor. The cofactor's pyridine ring is hydrogen bonded to Tyr225, Asn194, and Asp222 and is pi-stacked with the indole ring of Trp140. In addition, Arg292 and Arg386, which are H-bonded to the carboxylate groups of the inhibitor, and His143, Ser139 and one water molecule, which modulate the interaction of Asp222 with the pyridine nitrogen, were also included in the calculations. Transitions were calculated for the two states in which the pyridine nitrogen and Asp222 interact either as a neutral or ion pair. For each of these, the three alternatives considered were the protonated and deprotonated aldimine and the enolimine. Geometries were taken from the X-ray structural data with the H-atom positions determined using the AMI Hamiltonian or placed at standard bond length from the proton donor. A lognormal functional analysis of the calculated transitions of the six ionization states was carried out to construct a simulated solution spectrum. The lambda(max) of the bands resulting from this analysis showed good agreement with the experimental values (rms deviation of 6 nm), and the simulated spectrum exhibited a good fit to the observed spectrum. The fitting strongly suggests that at the pH of 7.5 of the measured solution spectrum, the pyridine nitrogen-Asp222 pair can be present in both the charged and neutral states. The plausibility of this result, which contradicts previous assumptions, is discuss, and electrostatic arguments are presented which support the tenability of this conclusion. The orientations of the transition dipole moments were calculated and compared with observed values.