Mechanistic insights into F₄₂₀-dependent glucose-6-phosphate dehydrogenase using isotope effects and substrate...

F₄₂₀-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F₄₂₀ cofactor to yield 6-phosphogluconolactone and the reduced cofactor, F₄₂₀H₂. Here, we aim to probe the FGD reaction mechanism using dead-end inhibition experiments, as well as solvent and substrate deuterium isotope effects studies. The dead-end inhibition studies performed using citrate as the inhibitor revealed competitive and uncompetitive inhibition patterns for G6P and F₄₂₀ respectively, thus suggesting a mechanism of ordered addition of substrates in which the F₄₂₀ cofactor must first bind to FGD before G6P binding. The solvent deuterium isotope effects studies yielded normal solvent kinetic isotope effects (SKIE) on k_cat and k_cat/K_m for both G6P and F₄₂₀. The proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed SKIE is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P-d₁ yielded KIE of 1.1 for both k_cat and k_cat/K_m, while the pre-steady state KIE on k_obs was 1.4. Because the hydride transferred to C5 of F₄₂₀ was the one targeted for isotopic substitution, these KIE values provide further evidence to support our previous findings that hydride transfer is likely not rate-limiting in the FGD reaction.