Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in the putP gene affect the kinetics of proline transport

Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of the putP gene, suggesting that they may replace amino acids at the active site of the enzyme. If putP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of three putP substrate specificity mutants. One class of mutation increases the K_m over 120-fold but only decreases the V_max fourfold. Such K_m mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases the V_max 80-fold without changing the K_m. V_max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both the K_m and V_max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na⁺/proline symport.