Kinetics of ternary complex formation between dihydrofolate reductase, coenzyme, and inhibitors

Abstract

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.

X-ray structure of the ternary MTX·NADPH complex of the anthrax dihydrofolate reductase: A pharmacophore for dual-site inhibitor design

“…Previously, the binding of MTX to E. coli DHFR was shown to decrease tryptophan fluorescence due to quenching of Trp 22 in ecDHFR (Appleman et al, 1988), and the same change in Trp fluorescence upon ligand binding has been observed by others (Dunn and King, 1980). In principal, binding at the substrate and the NADPH binding sites can be evaluated by fluorescence spectroscopy (Appleman et al, 1988).…”

Inhibitor Mediated Protein Degradation

“…TMP is a specific inhibitor of eDHFR with much lower affinity for mammalian DHFRs ( K d = 20 pM versus 4 μM for bacterial and mammalian DHFRs, respectively. Importantly, the interaction between eDHFR and TMP is noncovalent, though dissociation is very slow (T 1/2 ~20 minutes (Dunn and King, 1980)). TMP can be modified at the 4 position of the B ring with retention of potency and selectivity (Figure 2A; (Calloway et al, 2007)).…”

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

“…The rate constants for binding ligands to DHFR in either the cofactor or the substrate binding sites can be measured by following the quenching of the intrinsic enzyme fluorescence due predominantly to its tryptophan residues. In the formation of binary complexes of DHFR at saturating substrate concentration, two exponentials were observed, a rapid phase whose rate was dependent on ligand concentration followed by a ligand-independent phase, while in the formation of ternary complexes (by addition of a ligand to the binary complex) a single ligand-dependent exponential is observed (Dunn et al, 1978; 3.50 - Dunn & King, 1980;Cayley et al, 1981). Cayley et al (1981) concluded that this behavior is due to the mechanism shown in Scheme I, where substrate binds rapidly to only one of two conformers of the free enzyme and interconversion between conformers is slow.…”