The relatively small quantities of enzymes typically contained in cells hamper determination of their presence and abundance. However, the ability to rapidly transform thousands of molecules of a specific substrate into products imbues each enzyme with the ability to amplify its presence. Under appropriate conditions, the rate of the catalytic reaction being monitored is proportionate to the amount of enzyme present, which allows its concentration to be inferred. Assays of the catalytic activity of enzymes are frequently used both in research and clinical laboratories.

Single-Molecule Enzymology

 The limited sensitivity of traditional enzyme assays necessitates the use of a large group, or ensemble, of enzyme molecules in order to produce measurable quantities of product. The data obtained thus reflect the average-activity of individual enzymes across multiple cycles of catalysis. Recent advances in nanotechnology and imaging have made it possible to observe catalytic events involving discrete enzyme and substrate molecules. Consequently, scientists can now measure the rate of individual catalytic events, and sometimes a specific step in catalysis, by a process called single-molecule enzymology, an example of which is illustrated in Figure1.

Fig1. Direct observation of single DNA cleavage events catalyzed by a restriction endonuclease. DNA molecules immobilized to beads (blue) are placed in a flowing stream of buffer (black arrows), which causes them to assume an extended conformation. Cleavage at one of the restriction sites (orange) by an endonuclease leads to a shortening of the DNA molecule, which can be observed directly in a microscope since the nucleotide bases in DNA are fluorescent. Although the endonuclease (red) does not fluoresce, and hence is invisible, the progressive manner in which the DNA molecule is shortened (1→4) reveals that the endonuclease binds to the free end of the DNA molecule and moves along it from site to site.

Drug Discovery Requires Enzyme Assays Suitable for High-Throughput Screening

Enzymes are frequent targets for the development of drugs and other therapeutic agents. These generally take the form of enzyme inhibitors. The discovery of new drugs is greatly facilitated when a large number of potential pharmacophores can be simultaneously assayed in a rapid, automated fashion—a process referred to as high-throughput screening (HTS). HTS employs robotics, optics, data processing, and microfluidics to simultaneously conduct and monitor thousands of parallel enzyme assays. It thus serves as a perfect complement to combinatorial chemistry, a method for generating large libraries of chemical compounds spanning all possible combinations of a given set of chemical precursors.

Enzyme-Linked Immunoassays

 The sensitivity of enzyme assays can be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunosorbent assays (ELISAs) use antibodies covalently linked to a “reporter enzyme”, such as alkaline phosphatase or horse radish peroxidase, that can be used to produce easily-detected chromogenic or fluorescent products in vitro. Serum or other biologic samples to be tested are first placed in multi-well microtiter plates. Most proteins adhere to the plastic surface and thus are immobilized. Any exposed plastic that remains is subsequently “blocked” by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added and the antibodies allowed to adhere to any immobilized antigen molecules. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody is then determined by assaying the activity of the reporter enzyme, a quantity that should be proportional to the number of antibody molecules present and, presumably, the antigen molecules to which they are bound.

NAD(P)+-Dependent Dehydrogenases Are Assayed Spectrophotometrically

The physicochemical properties of the reactants in an enzyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the ability of a substrate or product to absorb light. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb light at a wavelength of 340 nm, whereas their oxidized forms NAD(P)+ do not (Figure 2). When NAD(P)+ is reduced, the absorbance at 340 nm therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, when a dehydrogenase catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 nm will be observed. In each case, the rate of change in absorbance at 340 nm will be proportionate to the quantity of the enzyme present.

Fig2. Absorption spectra of NAD+and NADH. Densities are for a 44-mg/L solution in a cell with a 1-cm light path. NADP+ and NADPH have spectra analogous to NAD+ and NADH, respectively.

The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is generally more difficult. In some instances, either the product or remaining substrate can be transformed into a more readily detected compound, although the reaction product may have to be separated from unreacted substrate prior to measurement. An alternative strategy is to devise a synthetic substrate whose product absorbs light or fluoresces. For example, hydrolysis of the phosphoester bond in p-nitrophenyl phosphate (pNPP), an artificial substrate molecule, is catalyzed at a measurable rate by numerous phosphatases, phosphodiesterases, and serine proteases. While pNPP does not absorb visible light, the anionic form of the p-nitrophenol (pKa 6.7) generated on its hydrolysis strongly absorbs light at 419 nm, and thus can be quantified.

Many Enzymes May Be Assayed by Coupling to a Dehydrogenase

Another quite general approach is to employ a “coupled” assay (Figure 3). Typically, a dehydrogenase whose substrate is the product of the enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction to which the dehydrogenase has been coupled.

Fig3. Coupled enzyme assay for hexokinase activity. The production of glucose-6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP+. When an excess of glucose-6-phosphate dehydrogenase is present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose-6-phosphate by hexokinase.

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