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rapidly frozen down as a concentrated stock to be stored in deep freeze (-20°C to -80°C). The freeze-thaw stability of an enzyme stock should be checked early on over several cycles and storage periods. In general, freezing and thawing should be as fast as possible, with gentle mixing to avoid the formation of pH and concentrations gradient in situ. In some cases, enzymes do not freeze well as solutions, so stabilizers such as BSA, gelatin, glycerol, sucrose, or cyclodex-trins can be added, or the enzyme can be stored as a salt pellet (e.g., ammonium sulfate) [25,26].

D. Substrate Effects on Initial Rates and Choice of Substrate

Knowledge of the Km of the substrate of an enzyme is necessary to set the concentrations desired in an assay. To find both competitive and noncompetitive inhibitors, one does not want to be too far above Km, subject to having enough substrate to give adequate rate and final signal. With soluble substrates it is often desirable to manipulate the assay conditions to bias the hits from screening to a particular binding site. If one does not want to miss competitive inhibitors, set the substrate concentration less than 3 times Km, so that the 1 + [S]/Km term does not get too large in the relationship for competitive inhibitors:

If, on the other hand, one wishes to exclude competitive inhibitors (e.g., molecules that bind at the ATP site of kinases) then one should set [S] > 10 X Km.

While most treatises of Michaelis-Menten kinetics use single substrate reactions, the vast majority of enzyme-catalyzed reactions involve more than one substrate. Figure 6 (curve a) is a typical rectangular hyperbolic plot of v versus [S], which is used to determine the Km of substrate. [S] is estimated by [S]tot, since usually [S] >> [E]. However, other methods such as a Dixon plot must be used to determine the Km (or Ki) of very high affinity substrates (or inhibitors), since the low range of concentrations used are often of the same magnitude as [E]tot [27]. It is worthwhile to point out that the assumptions of steady-state of all enzyme-substrate intermediates used to derive Michaelis-Menton kinetics fit experimental fact but do not prove mechanism. In fact, the Langmuir isotherm and chain reaction mechanism will give the same equations. Normalized concentration curves are rectangular hyperbolas, and the semilog plot looks like a classic IC50 [28] or Langmuir absorption isotherm [29]. For enzymes with more than one substrate the apparent Km of each substrate will vary, depending upon the saturation level of the other substrates. An estimate of one should be made at m

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