Enzymes advantages/disadvantages

There are other examples of HIV protease substrates that utilize the fluorescence energy transfer technique. Perhaps the strongest characteristic of these assays is that they provide a continuous readout of enzyme activity. Another advantage is their sensitivity (they use small concentrations of enzyme). The disadvantage of these assays is that they are susceptible to interference by some compounds (inhibition artifacts) because of inner and outer filter effects (see Notes 8-10). Other assays, for example, those based on radioactivity or high-performance liquid chromatography (HPLC) analysis of products are tedious to run, but are less susceptible to interference or inhibition artifacts. 

Enzyme/catalyst Reductant (auxiliary substance) Product Advantages Disadvantages Ref. 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

The variety of enzyme-catalyzed kinetic resolutions of enantiomers reported ia recent years is enormous. Similar to asymmetric synthesis, enantioselective resolutions are carried out ia either hydrolytic or esterification—transesterification modes. Both modes have advantages and disadvantages. Hydrolytic resolutions that are carried out ia a predominantiy aqueous medium are usually faster and, as a consequence, require smaller quantities of enzymes. On the other hand, esterifications ia organic solvents are experimentally simpler procedures, aHowiag easy product isolation and reuse of the enzyme without immobilization. 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

The major advantage associated with continuous assays is that the initial rate of product formation can be determined with complete confidence, and any unusual behavior of the enzyme would be immediately apparent. The major disadvantage is a question of throughput an instrument such as a platereader would remain dedicated to the reading of a single plate for the duration of the enzyme-substrate incubation period, compared with an equivalent discontinuous assay where an entire plate may be measured in a... 

Because ATP and GTP are required in many biosynthetic and mechanochemical reactions, the ability to manipulate their concentrations is particularly important in many kinetic studies. The three enzymes most commonly employed for this purpose are hexokinase, phosphofruc-tokinase, and alkaline phosphatase. Each of these enzymes offers advantages and disadvantages, and one must consider the design requirements for any particular experiment before using one of these to deplete ATP or GTP. 

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 159-162 activation effect, 63, 174, 175 analysis, 63, 140, 159-183 burst, 64, 20, 203, 215 enzyme concentration, 63, 175-177 hysteresis, 64, 197, 200-204 limitations, 63, 181-183 plotting, 63, 177-180 practical methods, 63, 175-177 reversible inhibitor action, 63, 163-175 reversible reaction, 63, 171-175 simulation of, 63, 180 advantages and disadvantages, 249, 61-62 analysis, in kinetic models of inhibition, 249, 168-169 concave-down, 249, 156 concave-up, 249, 156 with enzyme-product complex instability, 249, 88 with enzyme-substrate instabil-... 

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