Probing the active site of the reconstituted aspartate/glutamate carrier from mitochondria

Abstract

Treatment of the reconstituted aspartate/glutamate carrier from mitochondria with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (Nbd‐Cl) led to complete inactivation of carrier function. Inhibition could be attributed to chemical modification of one single cysteine in the active site. This residue was specifically protected in the presence of aspartate or glutamate, 50% substrate protection being observed at half‐saturation of the external binding site. The bifunctional reagent 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (DIDS) also modified the same cysteine and, in addition, an active‐site lysine identified previously [Dierks, T., Stappen, R., Salentin, A. & Krämer, R. (1992) Biochim. Biophys. Acta 1103, 13–24]. The proximity of the cysteine [Cys(a)] and the lysine residue was confirmed by a mutual exclusion of the respective reagents when added consecutively. By using a variety of reagents a further cysteine [Cys(b)] and probably a histidine residue could be discriminated from Cys(a) and the lysine. The applied reagents were classified according to functional and structural criteria. Class A reagents, like Nbd‐Cl, modified the active‐site Cys(a) thereby inhibiting the antiport function. Class B reagents, like HgCl2, reacted with both Cys(a) and Cys(b) leading to a conversion of the carrier from antiport to uniport function [Dierks, T., Salentin, A., Heberger, C. & Krämer, R. (1990) Biochim. Biophys. Acta 1028, 268–280]. DIDS at relatively high concentration (60 μM) also acted as a uniport inducer. Class C reagents finally, like pyridoxal phosphate or diethyl pyrocarbonate, modified the active‐site lysine or histidine, respectively, and blocked antiport and uniport activity. By testing the accessibility of the mentioned residues to the various reagents, when applied in different order, topological relationships could be elaborated indicating the location of these amino acids with respect to the exofacial active site of the carrier protein.