Probing conformational states of spin-labeled aspartate aminotransferase by ESR.
Mitochondrial aspartate aminotransferase was selectively labeled with various maleimide-linked nitroxide spin labels at the conformationally sensitive Cys166. The mobility of the spin group was found to increase with increasing length of the spacer between the nitroxide and maleimide moiety. The label with the ethylcarbamoyl group, a spacer of intermediate length, responded sensitively to conformational changes of aspartate aminotransferase. The modification with this label decreased the enzymic activity to 30% of its initial value and increased the affinity for various substrates and inhibitors 5-10-fold. Identical ESR spectra were obtained for the pyridoxal and pyridoxamine form of the enzyme. These spectra are complex, consisting of an isotropic and at least two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local protein environment giving rise to different motional states. The same changes in the ESR spectra have been observed upon formation of the adsorption complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. Essentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptation of aspartate aminotransferase. The ESR data are consistent with an equilibrium between two conformational states of the enzyme but inconsistent with individual protein conformations of the various intermediates of the transamination reaction. The two conformational states may be assigned to the open and closed conformations as defined by X-ray crystallography. In the adsorption complex of the pyridoxal enzyme, and in the covalent intermediates, the two-state equilibrium appears to be shifted towards the closed conformation in which the spin label is more rigidly bound, as also suggested by molecular dynamic simulations of the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and aspartate or maleate was not accompanied by the same shift of the conformational equilibrium.
Sterk
M
M
Biochemisches Institut, Universität Zürich, Switzerland.
Hauser
H
H
Marsh
D
D
Gehring
H
H
D001219:Aspartate Aminotransferases / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D008301:Maleimides; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D011487:Protein Conformation; D013113:Spin Labels
D008301:Maleimides; D013113:Spin Labels; C002745:nitroxide maleimide; D001219:Aspartate Aminotransferases