title: A balancing act between net uptake of water during dihydrofolate binding and net release of water upon NADPH binding in R67 dihydrofolate reductase.
abstract: 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.
authors: Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
authors: Shaileja
authors: S
authors: Chopra
authors: Russell M
authors: RM
authors: Dooling
authors: Caroline Glyn
authors: CG
authors: Horner
authors: Elizabeth E
authors: EE
authors: Howell
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000737:chemistry / Q000494:pharmacology; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D009995:Osmosis / Q000187:drug effects / Q000502:physiology; D010084:Oxidation-Reduction / Q000187:drug effects; D020836:Protein Structure, Quaternary; D013012:Sorbitol / Q000494:pharmacology; D013549:Sweetening Agents / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D014295:Trimethoprim / Q000737:chemistry / Q000494:pharmacology; D014867:Water / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D005493:Folic Acid Antagonists; D013549:Sweetening Agents; D014867:Water; C010920:dihydrofolate; D013012:Sorbitol; D009249:NADP; D005492:Folic Acid; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase