The Concept and Determination of Enzyme Activity

If the course of the reaction is followed, a curve like the one depicted in Fig 1.4 will be obtained. 

This means that the reaction rate (slope of the p vs t curve) will decrease as the reaction proceeds. Then, the use of Eq. 1.1 is ambiguous if used for the determination of enzyme activity. To solve this ambiguity, the reasons underlying this behavior must be analyzed. The reduction in reaction rate can be the consequence of desaturation of the enzyme because of substrate transformation into product (at substrate depletion reaction rate drops to zero), enzyme inactivation as a consequence of the exposure of the enzyme to the conditions of reaction, enzyme inhibition caused by the products of the reaction, and equilibrium displacement as a consequence of the law of mass action. Some or all of these phenomena are present in any enzymatic reaction so that the catalytic capacity of the enzyme will vary throughout the course of the reaction. It is customary to identify the enzyme activity with the initial rate of reaction (initial slope of the p versus t curve) where all the above mentioned 

This is actually a very convenient method for determining activity of such class of enzymes, since organic coenzymes (i.e. FAD or NADH) are usually very easy to determine analytically. An example of a coupled system considering coenzyme determination is the assay for lactase ((3-galactosidase EC 3.2.1.23). The enzyme catalyzes the hydrolysis of lactose according to  

Glucose produced can be coupled to a classical enzymatic glucose kit, that is hex-oquinase (Hx) plus glucose 6 phosphate dehydrogenase (G6PD), in which  

Enzyme activity can be determined by a continuous or discontinuous assay. If the analytical device is provided with a reeorder that register the course of reaction, the initial rate could be easily determined from the initial slope of the product (or substrate, or coupled analyte, or coenzyme) concentration versus time curve. It is not always possible or simple to set up a continuous assay in that case, the course of reaction should be monitored discontinuously by sampling and assaying at predetermined time intervals and samples should be subjected to inactivation to stop the reaction. This is a drawback, since the enzyme should be rapidly, completely and irreversibly inactivated by subjecting it to harsh conditions that can interfere with the 

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