Enzymes, Genetic Manipulation Of
The primary goal of genetic manipulation and design of enzymes is to create enzymes that possess some improved or novel activities and properties. These modifications can provide a broader understanding of how an enzyme's structure relates to its function, what changes can be introduced in order to expand the use of enzymes in biotechnology or medicine. Genetically manipulated enzymes have been created through exchange of aminoacids or modifications of secondary and tertiary structures yielding enzymes with novel substrate specificity, biophysical stability, and catalytic properties. Genetic manipulation may involve altering the many regions or activities of enzymes including catalytic, noncatalytic and regulatory properties.
Alteration of noncatalytic and regulatory properties may involve the insertion of short peptide domains with defined functions. These domains may serve in stabilizing the enzyme, targeting it to a specific compartment or substrate like the cell membrane or DNA. These peptide domains could also be used as protein tags to facilitate the purification of the enzyme by special affinity chromatography. In some cases, the insertion of an epitope, the binding site of an antibody, in a regulatory domain close to the enzyme active site may allow the modulation of that enzyme by the antibodies specific for that epitope.
The most commonly used genetic modifications aim at the creation of enzymes with novel catalytic activities and specificities. Most of the alterations involve introducing point mutations, exchanging protein domains, and modifying secondary and/or tertiary structures. All these manipulations result in the modulation of the binding sites, affinity for substrates, and susceptibility for inhibition. Point mutations have been used successfully to introduce changes in the substrate specificity of many proteases. Many bacterial enzymes have thus been mutated to closely mimic the enzyme activity of their eukaryotic counterparts. In other cases, enzyme modifications through point mutations led to conversion in preferences for coenzymes and to changes in enzyme kinetics (the study of the steps and rates of reaction for an enzyme).
Novel genetic modification strategies that aim at fine-tuning the activation and inhibition of enzymes include random modifications through point mutations that only change the inhibition susceptibility of the enzyme while keeping its catalytic specificity intact. The mutagenesis is followed by selection using small, specific and strong inhibitors of the novel mutant enzymes. This strategy, which has so far been tested on members of the large family of protein kinases, is very promising because it allows the characterization of enzymes from the sub-molecular level (an amino-acid modification in a domain of the enzyme) to application in the in vivo model of a whole organism. This strategy promises to allow the design of a new generation knock-out animal models where the targeted gene is normally functioning in the animal model (because the active site of the enzyme is intact) until the specific inhibitor is added. The strong and specific inhibition thus obtained gives an effect on the whole animal very similar to that of a knock-out by gene disruption. An advantage is that the animal model that carried this kind of mutation is not altered to the same extent as the animal that had an inactive enzyme from the early embryonic stage. Furthermore this model gives the investigating scientist the possibility to study the effect of removal of an enzyme activity from the whole organism even at a very narrow time window (Something that is very difficult to achieve with conventional knock-out models).
Besides introducing changes by point mutation, enzymes can be modified by exchange of whole functional domains or protein modules. This approach usually involves the engineering of fusion proteins and enzyme hybrids. Such an approach is usually chosen if one has sufficient knowledge about the specificity of an enzyme but want to target that enzyme activity to a specific site. Some DNA restriction enzymes especially the type-II restriction endonucleases, which act through distinct DNA binding and cleavage domains, have been designed in this way.
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