Enantioselective enzymes reactions, assaying

Fig. 7. Enzyme-coupled assay in which the hydrolase-catalyzed reaction releases acetic acid. The latter is converted by acetyl-CoA synthetase (ACS) into acetyl-CoA in the presence of (ATP) and coenzyme A (CoA). Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to give citrate. The oxaloacetate required for this reaction is formed from L-malate and NAD in the presence of L-malate dehydrogenase (l-MDH). Initial rates of acetic acid formation can thus be determined by the increase in adsorption at 340 nm due to the increase in NADH concentration. Use of optically pure (Ry- or (5)-acetates allows the determination of the apparent enantioselectivity i app i81)-...

The advantage of fluorescence-based assays is their high sensitivity. It is therefore perhaps surprising that few such systems have been developed for evaluating the enantioselectivity of enzyme-catalyzed reactions. Fluorescence as a detection method is used in an enzyme-coupled assay [26] (see Section 9.3.4.3) and in the capillary array electrophoresis [25] (see Section 9.3.6.5). If several substrates need to be screened simultaneously, fluorescence-based substrate arrays as enzyme fingerprinting tools can be used, although enantioselectivity still needs to be addressed [26e],... 

Fluorescence-based assays either in the measurement of enzyme activity or in the quantification of enantioselectivity all have a high degree of sensitivity, which allows the use of very dilute substrate concentrations and extremely small amounts of enzymes. Basically, there are two different approaches. One involves the use of a substrate of interest to which a fluorescent-active (or potentially active) moiety is covalently attached. The second approach makes use of a fluorescence-based sensor, which gives rise to a signal as a consequence of the enzyme-catalyzed reaction of a substrate of interest. 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

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