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  • Repulsive interactions towards undesirable substrates are ar


    Repulsive interactions towards undesirable substrates are arguably a very efficient means to implement specificity [8]. In particular, it could be assumed that discrimination against a substrate that is larger than the cognate substrate may be achieved easily by restricting the active site and exploiting steric repulsion [12]. However, lenvatinib are relatively flexible (and there is experimental evidence that flexibility may correlate with substrate promiscuity; e.g., 20, 21, 22) while active sites must have an opening towards the solvent; it has been suggested that such features in specific cases may allow conversion of bulkier alternative substrates [9]. In this instance the actual limits to specificity are nearly impossible to estimate a priori. Figure 1C examines a practical case, considering how enzymes that act on a linear substrate discriminate against a competitor metabolite containing one additional methylene group. In this case too, many enzymes show relatively modest selectivity. Note that alternative activities may occur even with substrates that are much larger than the canonical substrate. For example, it was shown that several mammalian transaminases, whose standard substrates are simple amino acids, can transaminate the tripeptide glutathione with low efficiency [23].
    Detrimental Activities with Alternative Substrates May Be Redressed by Repair Enzymes An opposite scenario arises when the activity of a metabolic enzyme with an alternative substrate is seriously detrimental to fitness. In fact, every such reaction may generate a product that is useless for the cell (a metabolic dead-end, implying a waste of resources) or that is even toxic [44], and this is expected to elicit strong evolutionary pressure to improve the specificity of the enzyme. However, as discussed, there are limits to such an improvement. Preventing access of the enzyme to the alternative substrate by compartmentalization may also be often impossible or insufficient. Instead, evolution can lead to the development of ad hoc enzymes that destroy or recycle the unwanted products of these side reactions. In fact, there is a growing list of ‘metabolite repair’ enzymes whose sole purpose appears to be the correction of ‘errors’ committed by enzymes of intermediary metabolism 45, 46, most often attributable to imperfect substrate specificity. These resemble the proofreading activities that improve the accuracy of aminoacyl-tRNA synthesis [45]; the repair task is sometimes even allocated to a separate domain of the promiscuous metabolic enzyme, not unlike what happens in many aminoacyl-tRNA synthetases [47]. One exemplary case is the activity of malate dehydrogenase with α-ketoglutarate instead of with the standard substrate oxaloacetate. The relative efficiency of the promiscuous reaction is very low, and the discrimination index of malate dehydrogenase is estimated to be ∼106[48], in other words higher than for any of the enzymes in Figure 1C. Despite this, the alternative reaction is not physiologically insignificant because both α-ketoglutarate and malate dehydrogenase are abundant in cells, whereas L-2-hydroxyglutarate is a metabolic dead-end, which favors its accumulation over time. Normally, such accumulation is prevented by a dedicated repair enzyme, a FAD-dependent dehydrogenase that irreversibly reoxidizes L-2-hydroxyglutarate to α-ketoglutarate. The fact that, in humans, a deficiency of the repair enzyme causes L-2-hydroxyglutaric aciduria (a severe neurological disorder) [49] underscores the potential dangers of even slow side-activities of substrate-promiscuous enzymes. Similarly, the slow transamination of glutathione carried out by many transaminases (mentioned earlier) generates an apparently useless product, deaminated glutathione. This compound, however, can be hydrolyzed and recycled by a ‘repair’ amidase that has been identified in mammals, yeast, and several glutathione-producing bacteria [23]. It is notable that the compound processed by this amidase appears to originate from the side activities of an entire class of enzymes. This attests to the efficiency of metabolite repair as an evolutionary solution to the inherent imperfection of metabolic catalysts. An analogous and even more striking example is that of a ‘repair’ phosphatase that degrades inhibitory compounds generated by the substrate-promiscuous activities of two glycolytic enzymes – glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase (Figure 2) [46].