Binding of coenzyme analogs to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes

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Abstract
The binding, or association constants of NADP+, NADPH and a series of structural analogs to dihydrofolate reductase from L. casei MTX/R were determined fluorometrically. Modification of the adenine or nicotinamide rings has little effect on the binding of the oxidized coenzyme, but the thionicotinamide and acetylpyridine analogs of the reduced coenzyme bind much more weakly than NADPH itself. In the presence of the substrate, folate, or the inhibitors methotrexate or trimethoprim, the oxidized coenzymes bind appreciably more tightly to the enzyme. The magnitude of this cooperativity, which covers a range of 1- to 37-fold, depends markedly on the structure of both the coenzyme and the substrate or substrate analog; 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 others. The presence of the inhibitors leads to very substantial decreases in the coenzyme dissociation rate constant.sbd.by factors of 300-2200. The binding constant of methotrexate in the ternary complex is .apprx. 1.3 .times. 1012 M-1. The structural origins of the differences in binding constant and cooperative behavior of the various coenzymes and coenzyme analogs are discussed in the light of information from crystallography and NMR spectroscopy.