The effects of modification with N-bromosuccinimide on the binding of ligands to dihydrofolate reductase.
The binding constants of substrate, inhibitors and coenzymes to native Lactobacillus casei dihydrofolate reductase and to the enzyme modified (at Trp-21) by N-bromosuccinimide have been determined using fluorimetric and spectrophotometric methods. The modification leads to only modest decreases (factors of 2-4) in the binding of substrate or substrate analogues, but the effects of coenzyme binding are much larger. The binding of NADPH is decreased by a factor of 200, but that of NADP+ by only a factor of 4, indicating a clear difference in their mode of interaction with the enzyme. The nature of this difference is discussed in the light of crystallographic and n.m.r. studies of the enzyme.
D001974:Bromosuccinimide; D003067:Coenzymes; D005493:Folic Acid Antagonists; D007700:Kinetics; D008024:Ligands; D009249:NADP; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013388:Succinimides; D013762:Tetrahydrofolate Dehydrogenase
D003067:Coenzymes; D005493:Folic Acid Antagonists; D008024:Ligands; D013388:Succinimides; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D001974:Bromosuccinimide