Fig. 4.

Altered substrate specificity for OxlT variants exposed to MTSEA. To determine apparent inhibition constants, the initial rates of 0.1 mM [¹⁴C]oxalate transport were determined in the presence of malonate (panel A), formate (panel B) or nitrate (panel C). Proteoliposomes treated with 1 mM MTSEA contained either cysteine-less OxlT (open circles), or its K355C (open squares) or R272C (closed circles) derivative. Observed rates were normalized by expressing them as a fraction (F) of the rate found in the absence of inhibitor, and apparent K_i values (K_iapp) were determined as the inhibitor concentrations yielding 50% inhibition of [¹⁴C]oxalate transport. K_iapp values cited in the text summarize three independent experiments as shown here. Assuming simple competitive inhibition and the kinetic parameters in Table I, the observed K_iapp values for malonate would reflect true K_i values of 72 mM, 53 mM, and 2.7 mM for R272C_MTSEA, K355C_MTSEA and cysteine-less OxlT, respectively; for formate, the respective K_i values would be 0.31 mM (K355C_MTSEA) and 0.71 mM (cysteine-less OxlT); for nitrate, these K_i values would correspond to 0.7 mM (K355C_MTSEA) and 1.8 mM (cysteine-less OxlT).