Autonomous folding and coenzyme binding of the excised pyridoxal 5'-phosphate binding domain of aspartate aminotransferase from Escherichia coli.
The coenzyme (PLP) binding domain (residues 47-329) of the dimeric aspartate aminotransferase from Escherichia coli was produced separately by recombinant DNA methods. It folded autonomously both in vivo and in vitro, that is, independently of the native N- and C-terminal extensions that combine to form the small domain of eAAT. The PLP-domain had one binding site for PLP of relatively high affinity involving a covalent bond to the protein. It was monomeric, although the major subunit-subunit interface at the 2-fold symmetry axis remained unchanged. This effect appears to be due mainly to the absence of the N-terminal extension that contains hydrophobic residues, which interact with the PLP-domain of the second subunit in the wild-type dimer. Judged by circular dichroism, fluorescence, and HPLC gel filtration at increasing concentrations of guanidinium chloride, the PLP-domain underwent a three-state unfolding transition (M' in equilibrium M'* in equilibrium U') involving a compact intermediate M'*. This behavior parallels the unfolding of the dissociated native monomer of cAAT.
Herold
M
M
Abteilung für Biophysikalische Chemie, Universität Basel, Switzerland.
Leistler
B
B
Hage
A
A
Luger
K
K
Kirschner
K
K
D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry / Q000235:genetics; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism; D007097:Imines / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D014961:X-Ray Diffraction
D001051:Apoenzymes; D003067:Coenzymes; D006146:Guanidines; D007097:Imines; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases; D019791:Guanidine