Substrate cycles compartmentation

Tawfik and Griffith (1998) reported an in vitro selection strategy for catalytic activity using compartmentalization. Here, each member of the DNA library is encapsulated in an aqueous compartment in a water in oil emulsion. The compartments are generated from an in vitro transcription-translation system, and contain the components for protein synthesis. The dilution is chosen such that, on average, the water droplets contain less than one DNA molecule. The DNA is transcribed and translated in vitro in the presence of substrate, which is covalently attached to the DNA. Only translated proteins with catalytic activity convert the substrate to the product. Subsequently, all DNA molecules are recovered from the water droplets and the DNA linked to the product is separated from the unmodified DNA linked to the educt, which requires a method to discriminate between both. The modified DNA can then be amplified by PCR and used for a second selection cycle. The principle of this approach is depicted in Figure 6. 

In the cell, compartmentation of enzymes into multienzyme complexes or organelles provides a means of regulation, either because the compartment provides unique conditions or because it limits or channels access of the enzymes to substrates. Enzymes or pathways with a common function are often assembled into organelles. For example, enzymes of the TCA cycle are all located within the mitochondrion. The enzymes catalyze sequential reactions, and the product of one reaction is the substrate for the next reaction. The concentration of the pathway intermediates remains much higher within the mitochondrion than in the surrounding cellular cytoplasm. 

In a review of their work, Caplan and Naparstek pointed out that a simpler system might have been an enzyme-free membrane separating the alkahne BAEE solution from a small chamber containing the papain [56]. The chamber could be treated as homogeneous, and quasi-steady-state ordinary differential equations could account for transport of substrate, acid, and base across the membrane as well as for the enzyme-catalyzed reaction. By fixing the external concentrations of BAEE and H+ (and hence OH since the dissociation product is constant), it was shown that conditions exist under which diffusional and reaction fluxes that balance each other are unstable, and the system is directed to a hmit cycle. This simplified membrane-chamber system was further investigated theoretically by Ohmori et al [59], who identified regions of parameter space that are predictive of pH oscillations for compartmentalized papain and other proteolytic... 

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