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-Å x-ray diffraction data to an R value of 0.170, and with 1.6-Å data to an R value of 0.173. In the presence of 2-methylaspartate, one of the subunits (subunit 1) shows a ligandinduced 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 (,) 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.

Anion Recognition in Water: Recent Advances from a Supramolecular and Macromolecular Perspective

“…c) Simplified illustration of the C α NN motif. d) Binding of a phosphate group by a C α NN motif located at the end of a helix (PDB 1AJS57). …”

Crystal Structure of Human Kynurenine Aminotransferase II

“…At the same time, the pyridine ring of PMP also tilts toward the substrate and leaves the C4 atom vulnerable for substrate attack. It is well known that by binding substrates, aspartate aminotransferase changes its conformation from an open to a closed form (61)(62)(63)(64)(65)(66), which involves a largescale conformational change (domain-domain rotation). In hKAT-II, however, there is no large scale conformational change upon binding of the substrate.…”

Pre-steady-state Reaction of 5-Aminolevulinate Synthase

“…These steps could represent an initial noncovalent interaction followed by slow conversion of the internal aldimine to the external aldimine or formation of the external aldimine followed by a slow conformational change of the enzyme, among other possibilities. Many PLP-dependent enzymes, including aspartate aminotransferase (22), serine hydroxymethyltransferase (23), and tryptophan synthase (24), undergo transitions from "open" to "closed" conformations upon amino acid binding. Differences in the two conformations have been described in detail for aspartate aminotransferase (22,(25)(26)(27)(28).…”

“…Many PLP-dependent enzymes, including aspartate aminotransferase (22), serine hydroxymethyltransferase (23), and tryptophan synthase (24), undergo transitions from "open" to "closed" conformations upon amino acid binding. Differences in the two conformations have been described in detail for aspartate aminotransferase (22,(25)(26)(27)(28). Closure of the enzyme around the substrate appears to function primarily to increase substrate specificity.…”