Refinement and comparisons of the crystal structures of pig cytosolic aspartate aminotransferase and its complex with 2-methylaspartate

Two high resolution crystal structures of cytosolic aspartate aminotransferase from pig heart provide additional insights into the stereochemical mechanism for ligand-induced conformational changes in this enzyme, Structures of the homodimeric native structure and its complex with the substrate analog 2-methylaspartate have been refined, respectively, with 1.74-Angstrom x-ray diffraction data to an R value of 0.170, and with 1.6-Angstrom data to an R value of 0.173, In the presence of 2-methylaspartate, one of the subunits (subunit 1) shows a ligand induced conformational change that involves a large movement of the small domain (residues 12-49 and 327-412) to produce ''closed'' conformation, No such transition is observed in the other subunit (subunit 2), because crystal lattice contacts lock it in an ''open'' conformation like that adopted by subunit 1 in the absence of substrate, By comparing the open and closed forms of cAspAT, we propose a stereochemical mechanism for the open-to-closed transition that involves the electrostatic neutralization of two active site arginine residues by the negative charges of the incoming substrate, a large change in the backbone (phi,psi) conformational angles of two key glycine residues, and the entropy-driven burial of a stretch of hydrophobic residues on the N-terminal helix, The calculated free energy for the burial of this ''hydrophobic plug'' appears to be sufficient to serve as the driving force for domain closure.