DIFFERENTIAL SENSITIVITY OF STILBENEDISULFONATES IN THEIR REACTION WITH BAND-3 HT (PRO-868 -] LEU)

Band 3 HT (Pro-868 --> Leu) is a mutant anion exchange protein which h as several phenotypic characteristics, including a 2- to 3-fold larger V-max, and reduced covalent binding of the anion transport inhibitor ,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have u sed fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-ty pe protein. In contrast, no such difference in the kinetics was observ ed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonat e (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT w as abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disul fonate (DBDS) release showed no difference when compared with the wild -type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of ''lysine A'' in the me mbrane-bound domain of band 3 but rather produces an apparently locali zed conformational change in the C-terminal subdomain of the protein w hich alters H2DIDS affinity. When combined with the observation of an increased V-max, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.