In cells, enzymes often exist in multiple forms arising from the

In cells, enzymes often exist in multiple forms arising from the same (splice variants) or different loci in the genome. In contrast, in a reconstituted selleck kinase inhibitor biochemical system, the enzyme is often isolated, lacking many or all of its native binding partners, which can significantly affect enzyme stability and activity in vitro. Additionally, it may be difficult to express and purify the enzyme in its native form due to size and/or

stability limitations in the absence of these partner proteins. Hence, numerous constructs are often attempted in the expression and purification of the desired enzyme, including truncated variants and alternatively tagged species in an attempt to maximize protein yield, stability and activity. A caveat of these artificial alterations is that the more one diverges from the natural protein, the more likely it is to identify

compounds that lack a physiologically relevant mechanism and to miss compounds that work under physiological conditions. The choice of which protein construct to employ for development of the enzyme assay depends on several factors. Initial tests that assess differences in both the activity and stability of protein constructs are critical in deciding which constructs to advance. In addition, the use of “tool” compounds, that is compounds with known modes of inhibition (MoI), can be extremely revealing in evaluating which construct to ultimately use in a HTS ZD1839 based on the desired MoI. When multiple constructs of an enzyme use the same substrate, it is possible to compare their activities using the Michaelis–Menten

constants in the form of kcat/Km. This takes into account both the rate determining step which limits the maximal velocity of the enzyme reaction (expressed in the constant kcat) and the propensity of the substrate to be turned over to product (Km). While subtle differences in the rate and or Km may exist among constructs, large differences in kcat/Km can indicate significant differences in the structure and/or stability of the construct. Additionally, the specific activity can also be used to compare different preparations of the same enzyme construct. The specific activity is the Vmax (calculated at saturating amounts of substrate) divided by the mass of enzyme in the reaction and is usually expressed in μmol min−1 mg−1. A severe decrease in assay performance may be indicative of poor batch reproducibility or indicate a decay in batch activity which can be checked by monitoring the specific activity between different enzyme batches or different samples of enzymes from the same batch. The purity of the enzyme target must also be considered, as the use of an impure enzyme preparation can lead to the selection of aberrant or mis-targeted inhibitor compounds. Enzyme purity can be assessed in a number of ways.

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