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  • br When Does Activity with Alternative


    When Does Activity with Alternative Substrates Depend on Neutral Drift? In a final scenario, the alternative reaction catalyzed by an enzyme might have no significant (positive or negative) effects on the system fitness, and thus it would be invisible to natural selection and essentially subject to neutral drift. This is often assumed as the ‘default’ case [11], but positive proof is scarce. We have seen above that some reactions which, based on the discrimination factor, would appear very negligible, do become liabilities because the promiscuous enzyme and the alternative substrate are abundant in the cell. As a minimum, an estimate of the biological significance of the alternative reaction (and hence of the selective pressure to which it is subject) should take into account the effective rate of its occurrence in vivo. In addition, one may think that secondary activities that generate mainstream metabolites should be essentially irrelevant for fitness, and therefore depend on neutral drift. Perhaps the best case in point comes from a study by Khanal et al.[50], who analyzed the ability of nine microbial γ-glutamylphosphate reductases (ProA) to use the alternative substrate N-acetyl-glutamyl phosphate (which is usually processed by a distinct reductase, ArgC). The discrimination indices for the nine enzymes were always very high (>40000) while the absolute efficiency of the secondary activity varied up to 50-fold between species. Furthermore, the enzyme from E. coli could not compensate for the loss of ArgC. All these features were consistent with the side activity of ProA being biologically irrelevant [50]. In Efavirenz to the ProA case, however, enzymes whose secondary activities are metabolically redundant, and which can under some circumstances surrogate the function of more specialized catalysts, are often observed (e.g., 51, 52), and it cannot be excluded that the non-strict substrate specificity of these enzymes may be retained because it is beneficial. Speaking more generally, there are suggestions that side activities generating mainstream metabolites may provide advantages or disadvantages under particular conditions and therefore be under active selection [44]. For example, consider an aminotransferase whose primary substrate is not alanine. It might be assumed that a side activity on alanine may not be strongly favored or unfavored by evolution because the product, pyruvate, is a standard metabolic intermediate. However, the alternative reaction can contribute to metabolic redundancy, which may increase fitness under particular circumstances while ensuring the resilience of the metabolic system. In E. coli, for example, there are three major alanine aminotransferases plus up to seven other enzymes with substantial activity towards alanine. Such a remarkable redundancy has been proposed to be important to ensure a supply of D-alanine for peptidoglycan synthesis [53]. Conversely, there may be significant counterselection against side activities that, despite generating standard metabolites, represent an objective waste of energy or resources. An example could be the transamination of glutamine, which tends to be reserved to processes that need to be metabolically irreversible [54]. Another case worth considering is that of kinases and ligases. Many of these enzymes show a strong specificity for ATP, while others can use different nucleoside triphosphates (NTPs) with comparable efficiencies (e.g., 55, 56). These different behaviors could be the random results of distinct evolutionary histories, but again positive selection for different levels of specificity cannot be ruled out a priori. ATP is usually the most abundant NTP, and the activity of enzymes strictly dependent on ATP will therefore respond more promptly to the energy status of the cell, and this may be important in the perspective of regulating metabolic fluxes. On the other hand, kinases or ligases that are less selective may allow an organism to scavenge alternative NTPs depending upon their availability, thus responding to the need to preserve the efficiency of an important reaction under diverse metabolic conditions 55, 56.