A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase
| Autor: | Shaileja Chopra, Elizabeth E. Howell, Russell M. Dooling, Caroline Glyn Horner |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Osmosis
Biochemistry Catalysis Trimethoprim chemistry.chemical_compound Folic Acid Drug Resistance Bacterial Dihydrofolate reductase Escherichia coli Sorbitol Protein Structure Quaternary Molecular Biology chemistry.chemical_classification Binding Sites biology Escherichia coli Proteins Water Active site Isothermal titration calorimetry Cell Biology Tetrahydrofolate Dehydrogenase Enzyme chemistry Osmolyte Sweetening Agents biology.protein Folic Acid Antagonists NADPH binding Oxidation-Reduction NADP Homotetramer |
| Zdroj: | Journal of Biological Chemistry. 283:4690-4698 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m709443200 |
| Popis: | R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH.DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|