Binding of coenzyme analogues to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes.
The binding, or association, constants of NADP+ NADPH, and a series of structural analogues to dihydrofolate reductase from Lactobacillus casei MTX/R have been determined fluorometrically. Modification of the adenine or nicotinamide rings has little effect on the binding of the oxidized coenzyme, but the thionicotinamide and acetylpyridine analogues of the reduced coenzyme bind much more weakly than NADPH itself. In the presence of the substrate, folate, or the inhibitors methotrxate or trimethoprim, the oxidized coenzymes bind appreciably more tightly to the enzyme. The magnitude of this "cooperativity", which covers a range of 1-37 fold, depends markedly on the structure of both the coenzyme and the substrate or substrate analogue; the nicotinamide ring of the coenzymes is clearly important in these effects. The binding constants of the reduced coenzymes in the presence of methotrexate or trimethoprim were too high to be measured fluorometrically. The dissociation rate constants of the coenzymes from their ternary complexes were therefore measured and compared with the values for the binary complexes reported by Dunn and co-workers [Dunn, S. M. J., Bathchelor, J. G., & King, R. W.(1978) Biochemistry 17, 2356]. The presence of the inhibitors leads to very substantial decreases in the coenzyme dissociation rate constant--by factors of 300-2200. The binding constant of methotrexate in the ternary complex is calculated to be approximately 1.3 X 10(12) M-1. The structural origins of the differences in binding constant and cooperative behavior of the various coenzymes and coenzyme analogues are discussed in the light of information from crystallography and NMR spectroscopy.
D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008433:Mathematics; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase