Cellular substrate concentration, enzymes sensitivity

N is often limiting in the marine environment. Further, many enzymes are sensitive to cellular substrate concentrations rather than extracellular concentrations and it is difficult to measure the relevant intracellular metabohte pools. In vitro assays may affect the conformation of enzymes and the degree to which they are modified. For example, allosteric effects (see Section 1.3.3) may be modified under in vitro conditions. Many enzymes undergo posttranslational regulation wherein enzyme activity is affected by binding of activator/inactivator proteins and covalent modification of the enzyme (e.g., adenylylation, phosphorylation or carbamylation) (Ottaway, 1988). When there is posttranslational modification of enzymes, enzyme activity measured in assays may be unrelated to in vivo activity (see Section 2.2.1) and there are few ways to determine the extent of enzyme modification in nature. 

Another enzyme whose function is of central importance to histidine assimilation is the histidyl-tRNA synthetase. The quantity of this enzyme in the cell does not vary with the availability of histidine [19]. However, the activity of the enzyme probably is sensitive to cellular conditions. Studies of the purified synthetase have established the values of its substrates to be 140 nM for ATP, 26 nM for histidine, and 0.04 nM for tRNA" [20,21]. The intracellular concentration of histidine [18] is very close to its K value, so variations in its concentration should readily influence the activity of the synthetase. 

When diffusion of a substrate to an immobilized enzyme is relatively slow, the concentration of substrate is lower in the microenvironment than in the macroenvironment the resulting reduction in enzymic activity is referred to as diffusional inhibition, a factor attenuated by an inhibitor of the enzyme. Consequently, inhibition by products is more pronounced in the presence, than in the absence, of diffusional resistances at a given concentration of product in the macroenvironment. The anti-energistic interaction between chemical and diffusional inhibitions, however, makes the activity of a bound enzyme less sensitive to changes in product concentration in the macroenvironment. The interplay between the various types of product and diffusional inhibitions was illustrated graphically. Another consequence of the anti-energistic interaction is that the activity of the boimd enzyme at steady state is affected less by an inhibitor in the presence, than in the absence, of diffusion-limited transfer of the substrate. These effects also apply to membrane-bound enzymes in cellular milieu. 

The spatial control of cellular processes can also be a powerful tool in synthetic biology since the local concentration of enzymes and metabolites can have a great influence in the rate at which the biosynthetic reactions of the pathway occur (Medema et al 2011). Several natural systems use cellular compartmentalization as spatial controllers. The diffusion of small molecules in and out of the compartments is limited and selective, which can protect the pathway from competing reactions, increase substrate/intermediate concentrations and decrease product degradation (Shiue and Prather, 2012 Boyle and Silver, 2012 Parsons et al., 2010 Chessher et al., 2015). In addition, in eukaryotic systems this type of spatial control is frequently used to isolate sensitive metabolic processes from the rest of the cellular enviromnental (Shiue and Prather, 2012). For example, in plants the last biosynthetic steps of secondary metabolites are performed in vesicles to avoid selftoxicity (Sirikantaramas et al., 2008). In Salmonella enterica, specific-amino terminal sequences were found to direct proteins... 

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