title: Refinement and comparisons of the crystal structures of pig cytosolic aspartate aminotransferase and its complex with 2-methylaspartate.
abstract: 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-A x-ray diffraction data to an R value of 0.170, and with 1.6-A 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 a "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.
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: S
authors.initials: S
authors.lastname: Rhee
authors.forename: M M
authors.initials: MM
authors.lastname: Silva
authors.forename: C C
authors.initials: CC
authors.lastname: Hyde
authors.forename: P H
authors.initials: PH
authors.lastname: Rogers
authors.forename: C M
authors.initials: CM
authors.lastname: Metzler
authors.forename: D E
authors.initials: DE
authors.lastname: Metzler
authors.forename: A
authors.initials: A
authors.lastname: Arnone
mesh_terms: D019342:Acetic Acid / Q000494:pharmacology; D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000201:enzymology*; D005561:Formates / Q000494:pharmacology; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000031:analogs & derivatives* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013552:Swine
chemical_list: D005561:Formates; D046911:Macromolecular Substances; C030544:formic acid; D016202:N-Methylaspartate; C037699:2-methylaspartic acid; D001219:Aspartate Aminotransferases; D019342:Acetic Acid