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 after 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 Km over 120 fold but only decreases the Vmax fourfold. Such Km mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases the Vmax 80-fold without changing the Km. Vmax mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both the Km and Vmax of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport.