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Substrate inhibition by the aldehyde has been observed for decades in NAD(P)-dependent aldehyde dehydrogenase (ALDH) enzymes, which follow a Bi Bi ordered steady-state kinetic mechanism. In this work, by using theoretical simulations of different possible substrate inhibition mechanisms in monosubstrate and Bi Bi ordered steady-state reactions, we explored the kind and extent of errors arising when estimating the kinetic parameters and determining the kinetic mechanisms if substrate inhibition is intentionally or unintentionally ignored. We found that, in every mechanism, fitting of the initial-velocity data of apparently non-inhibitory substrate concentrations to a rectangular hyperbola produces important errors, not only in the estimation of V values, which were underestimated as expected, but, surprisingly, even more in the estimation of K values, which led to overestimation of the V/K values. We show that the greater errors in K arises from fitting data that do experience substrate inhibition, although it may not be evident, to a Michaelis-Menten equation, which causes overestimation of the data at low substrate concentrations. Similarly, we show that if substrate inhibition is not fully assessed when inhibitors are evaluated, the estimated inhibition constants will have significant errors, and the type of inhibition could be grossly mistaken. We exemplify these errors with experimental results obtained with the betaine aldehyde dehydrogenase from spinach, which show the errors predicted by the theoretical simulations and that these errors are increased in the presence of NADH, which in this enzyme favor aldehyde substrate inhibition. Therefore, we strongly recommend that substrate inhibition by the aldehyde be assessed in every ALDH kinetic study, particularly when inhibitors are evaluated. The common practices of using apparently non-inhibitory concentration range of the aldehyde, or of using a single high concentration of the aldehyde or of the coenzyme when varying the other to determine true kinetic parameters, should be abandoned.
This article was published in the following journal.
Name: Chemico-biological interactions
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