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Reduced nicotinamide adenine dinucleotide phosphate (NADPH), folate, dihydrofolate, and the inhibitors trimethoprim and methotrexate bind rapidly and reversibly to both dihydrofolate reductase isoenzymes isolated from Escherichia coli RT500. The coenzyme and substrates appear to bind to only one of the mixture of two forms of the isoenzyme present at equilibrium, while the inhibitors bind to both forms. The proportions of the two forms are different for the two isoenzymes and are pH dependent in each case. The measured association rate constants for substrates and inhibitors lie in the range (1--2) x 10(-7) M-1 s-1 at 25 degrees C but are unlikely to be diffusion controlled. The rate constant for NADPH binding is 2 x 10(6) M-1 s-1. The formation of binary complexes takes place through a multistep mechanism. A minimum of three steps is required to explain the kinetic results. An equilibrium between two or more forms of the enzyme--ligand complex governs the overall dissociation. The stability of this equilibrium is largely responsible for the tighter binding of inhibitors relative to substrate or coenzyme and also for the different binding strengths of inhibitors to the isoenzymes.

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The selectivity of benzylpyrimidines for bacterial dihydrofolate reductases was studied by using equilibrium and kinetic techniques. Trimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine] and a series of close structural analogues with different methoxy group substitutions on the benzyl showed in vitro Escherichia coli antibacterial activities that varied according to their degree of substitution. Trimethoprim, the most potent analogue tested, was 400-fold more active than benzylpyrimidine, and the monomethoxy and dimethoxy analogues were of intermediate antibacterial activity. The relative antibacterial potencies of all the compounds were directly proportional to their E. coli form 1 dihydrofolate reductase Ki values, as determined by classical enzyme kinetics.

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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.