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  • br Materials and methods br Results and discussion br

    2019-12-10


    Materials and methods
    Results and discussion
    Concluding remarks Together, the results of the theoretical kinetic simulations and of the analysis of the experimentally determined kinetic data of SoBADH performed in this work show that ignoring substrate inhibition causes potentially important errors in the determination of the kinetic parameters not only of the substrate that produce inhibition but also of the other substrate in bisubstrate reactions, as well as of inhibitors, which most likely could also be true for activators. And as a consequence, kinetic mechanisms may be grossly mistaken. Since ALDH Bufexamac are so prone to experience substrate inhibition by the aldehyde, we strongly recommend that, in order to properly determine their kinetic parameters and their kinetic mechanisms, the initial velocity experiments should be performed using a range of aldehyde concentrations as wide as practically possible. In other words, the possibility that a particular ALDH enzyme experiences substrate inhibition by the aldehyde should be fully explored before performing a thorough kinetic characterization, including the evaluation of inhibitors or activators, in order to observe if substrate inhibition occurs in this particular enzyme and then correctly design the experiments and fit the experimental initial velocity data to the appropriate equations. The common practice of using a concentration range of the substrate where apparently there is no inhibition by this substrate should be abandoned because, as we show in this work, the fitting process of these data not only produce errors in the estimation of the true Vmax values but also because it produces even greater errors in the estimation of Km values, and therefore errors in Vmax/Km. Likewise incorrect is the also common practice of purposely using a high concentration of the aldehyde when studying the saturation of the ALDH enzyme by NAD(P)+, or a high concentration of NAD(P)+ when studying the saturation by the aldehyde with the intention of ensuring saturation by the fixed substrate so that true kinetic parameters for either the coenzyme or the aldehyde could be determined from single saturation curves. But if the high aldehyde concentration is inhibitory, as it could easily be even if has not been previously detected in a saturation experiment using a limited range of aldehyde concentrations, the estimated kinetic parameters for the coenzyme will be wrong and misleading. In addition, a high concentration of the coenzyme increases the degree of substrate inhibition by the aldehyde and consequently could increase the errors in the determination of the kinetic parameters for the aldehyde. The extent of the errors depends on the degree of inhibition of a particular enzyme by a particular substrate, and although important they may not be quantitatively of much relevance in certain experiments—for instance when comparing the effects that a change in a critical residues has on the kinetic of the enzyme because of the usually great differences between the wild-type and the mutant enzymes. Even though, if ignored, these errors could lead to qualitatively wrong conclusions, particularly if the wild-type and mutant enzymes differ in their susceptibility to substrate inhibition.
    Conflicts of interest
    Acknowledgments The authors acknowledge the financial support of DGAPA, UNAM (PAPIIT grant IN220317), Consejo Nacional de Ciencia y Tecnología (CONACYT grants 252123 and 283524), and Faculty of Chemistry, UNAM (PAIP grant 5000-9119) to RAMC.
    Introduction Currently, several technique barriers hamper the industrial development of ethanol from biomass, and enzymatic hydrolysis is the well-known rate liming step (Dasari and Berson, 2007, Dasari et al., 2009, Dunaway et al., 2010, Nidetzky and Steiner, 1993, Valjamae et al., 1998, Ye and Berson, 2011). Many studies focused on studying chemical properties of cellulose that limit fast enzymatic conversion. However, these studies yielded contradicting results, as some findings suggested cellulose properties affected hydrolysis rate (Betrabet and Paralikar, 1977, Ooshima et al., 1983) while other results suggested cellulose properties did not affect hydrolysis (Lenz et al., 1990, Ohmine et al., 1983, Puls and Wood, 1991). Through “restart” experiment using new enzyme on partially hydrolyzed Avicel microcrystalline cellulose, it was found that reactivity of cellulose substrate did not change during the enzymatic hydrolysis process (Yang et al., 2006). Therefore, some factor other than the cellulose properties must limit the hydrolysis rate. It has been recently demonstrated using an AFM imaging technique that a “traffic jam” of cellobiohydrolase units on a substrate strip reduces hydrolytic efficiency (Igarashi et al., 2011). Our work also revealed that inactivation of adsorbed cellobiohydrolase1 (CBH1) is a major factor limiting the reaction rate (Ye and Berson, 2011, Ye, 2012).