19F-n.m.r. studies of 3',5'-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes.
19F-n.m.r. spectroscopy was used to study the binding of 3',5'-difluoromethotrexate to dihydrofolate reductase (tetrahydrofolate dehydrogenase) from Lactobacillus casei. The benzoyl ring of the bound difluoromethotrexate was found to 'flip' about its symmetry axis, and the rate (7.3 X 10(3) s-1 at 298 K) and activation parameters for this process were determined by lineshape analysis of the 19F-n.m.r. spectrum at a series of temperatures in the range 273-308 K. The contributions to the barrier for this process are discussed. Addition of NADP+ or NADPH to form the enzyme-difluoromethotrexate-coenzyme ternary complex led to an increase in the rate of benzoyl ring flipping by a factor of 2.6-2.8-fold, and to substantial changes in the 19F-n.m.r. chemical shifts. The possible nature of the coenzyme-induced conformational changes responsible for these effects is discussed.
D005493:Folic Acid Antagonists; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D005493:Folic Acid Antagonists; D046911:Macromolecular Substances; C040821:3',5'-difluoromethotrexate; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate