I. Miyahara et al., X-RAY CRYSTALLOGRAPHIC STUDY OF PYRIDOXAMINE 5'-PHOSPHATE-TYPE ASPARTATE AMINOTRANSFERASES FROM ESCHERICHIA-COLI IN 3 FORMS, Journal of Biochemistry, 116(5), 1994, pp. 1001-1012
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.