X-RAY CRYSTALLOGRAPHIC STUDY OF PYRIDOXAMINE 5'-PHOSPHATE-TYPE ASPARTATE AMINOTRANSFERASES FROM ESCHERICHIA-COLI IN 3 FORMS

The three-dimensional structures of pyridoxamine 5'-phosphate-type asp artate aminotransferase from Escherichia coli and its complexes with m aleate and glutarate have been determined by X-ray crystallography at 2.2, 2.1, and 2.7 Angstrom resolution, respectively. The enzyme is a d imeric form comprising two identical subunits, each of which is divide d into one large and one small domain. The complex with maleate showed that substrate (or inhibitor) binding induced a large conformational change from the ''open'' to the ''closed'' form, resulting in closure of the active site by the small domain movement, as was observed in th e pyridoxal 5'-phosphate-type enzyme. In the open form, three hydropho bic residues (hydrophobic plug) at the entrance of the active site are exposed to solvent. Maleate binding make the active site more hydroph obic by charge compensation and release of water molecules, facilitati ng the movement of the hydrophobic plug into the active site pocket to induce a large conformational change in the enzyme. Maleate is fixed rigidly in the active site pocket by extensive salt bridges and a hydr ogen bonding network, guaranteeing the stereo-specificity of the catal ysis and giving a Michaelis complex model. Contrary to our expectation , the glutarate complex was in the open form, suggesting that the equi librium between the open and closed forms lies far toward the open for m in solution. The water molecules located in the active site pocket w ere almost completely conserved between Escherichia coli and chicken m itochondrial aspartate aminotransferase with the same type of cofactor and the same conformation.