Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in theputP gene affect the kinetics of proline transport |
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Authors: | Richard S Myers David Townsend Stanley Maloy |
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Institution: | (1) Department of Microbiology, University of Illinois, 61801 Urbana, Illinois;(2) Present address: Institute of Molecular Biology, University of Oregon, 97403 Eugene, Oregon;(3) Present address: Department of Biological Sciences, Illinois State University, 61761 Normal, Illinois |
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Abstract: | Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP 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 threeputP substrate specificity mutants. One class of mutation increases theK
m
over 120-fold but only decreases theV
max fourfold. SuchK
m
mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV
max 80-fold without changing theK
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 theK
m
andV
max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport. |
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Keywords: | putP substrate specificity proline transport |
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