br Materials and methods br Results and discussion
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 Ivabradine HCl 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 The industrial use of enzymes has seen a significant rise in the last decade which is owed in a large extent to the ease with which enzymes can be genetically tailored [1,2]. Properties such as substrate scope, enantioselectivity, solvent tolerance and thermostability can be adapted via enzyme engineering leading to catalysts that have been improved on a multifactorial level . In the chemical and pharmaceutical industry, biocatalysts are most often used for the installation of chirality and the late stage functionalization of scaffolds while in the production of aroma compounds enzymatic catalyst confer the added benefit of the ‘natural’ label as defined by the European flavor and food legislation. Biocatalytic strategies for the chiral resolution of esters and the synthesis of enantiopure alcohols and amines are particularly well established and are successfully applied in the biocatalytic production of pharmaceuticals such as sitagliptin , montelukast  and cipargamin . Despite the increasing industrial and academic interest in harnessing enzymes for synthesis, our understanding of the structure–function relationship in proteins is still limited  and enzyme engineering is considered to be mostly a collection of case-studies . However, recent protein optimization campaigns shed more light on selected enzyme families and highlight that more generalized engineering principles can be elucidated [8,9].