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 increas e with increasing length of the spacer between the nitroxide and malei mide moiety. The label with the ethylcarbamoyl group, a spacer of inte rmediate length, responded sensitively to conformational changes of as partate aminotransferase. The modification with this label decreased t he enzymic activity to 30% of its initial value and increased the affi nity for various substrates and inhibitors 5-10-fold. Identical ESR sp ectra were obtained for the pyridoxal and pyridoxamine form of the enz yme. These spectra are complex, consisting of an isotropic and at leas t two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local prote in environment giving rise to different motional states. The same chan ges in the ESR spectra have been observed upon formation of the adsorp tion complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. E ssentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptatio n of aspartate aminotransferase. The ESR data are consistent with an e quilibrium between two conformational states of the enzyme but inconsi stent with individual protein conformations of the various intermediat es of the transamination reaction. The two conformational states may b e assigned to the open and closed conformations as defined by X-ray cr ystallography. 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 mo re rigidly bound, as also suggested by molecular dynamic simulations o f the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and as partate or maleate was not accompanied by the same shift of the confor mational equilibrium.