Kinetics of ternary complex formation between dihydrofolate reductase, coenzyme, and inhibitors.
The kinetics of ligand binding to dihydrofolate reductase from Lactobacillus casei (MTX/R) to form the ternary enzyme-inhibitor-coenzyme complex have been investigated by the stopped-flow fluorescence technique. The fluorescence changes observed when coenzymes or inhibitors bind to the binary complex of the enzyme with the complementary ligand occur in a single fast phase. Under pseudo-first-order conditions the reaction traces could be fitted with precision to a single-exponential decay, and apparent bimolecular rate constants in the range 2 x 10(6) to 3 x 10(7) M-1s-1 have been measured assuming a bimolecular-unimolecular model. The kinetic constants obtained suggest that prior binding of an inhibitor to the enzyme may, to a minor extent, interfere with coenzyme binding but the rates of inhibitor binding seem to be unaffected by the presence of a bound coenzyme. Dissociation rate constants appear to be less than 1 s-1 which suggests that both coenzymes and inhibitors are tightly bound in the ternary complex. An investigation of the effects of pH on the kinetics of ternary complex formation indicated the involvement of ionizable groups in ligand binding, but this shows some ligand dependence. The rates of ligand bindings to form the ternary complex are fairly high, but it is unlikely that these associations are diffusion controlled because their measured activation energies of 7.8-14.5 kcal mol-1 are higher than expected from reactions whose rates are limited by diffusion in aqeous solution.
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology; D009249:NADP / Q000031:analogs & derivatives*; D011485:Protein Binding; D011739:Pyrimethamine / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
D011743:Pyrimidines; D009249:NADP; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate; D011739:Pyrimethamine