Enzyme-substrate pairs

The diversity of the structures of naturally occurring sugar nucleotides and of their enzymic reactions has been described in this Chapter. The question now arises as to whether the recognition processes are unique for each enzyme-substrate pair, or whether there exist some common features between different enzymes in this respect. The latter possibility seems more attractive and, more importantly, it is supported by some experimental evidence. 

Km values for several enzyme-substrate pairs are given in table 7.1. The values vary over a wide range but typically lie between 1CT6 and 10 1 m. With enzymes that can act on several different substrates, Km can vary substantially from substrate to substrate. With the enzyme chymo-trypsin, for example, the Km for the substrate glycyl-tyrosinamide is about 50 times that for the substrate A-benzoyltyrosinamide. 

Rules selected for this reaction included the joining and breaking probabilities as described earlier. In addition, it was necessary to include a probability of conversion, Pc, of an enzyme-substrate pair, ES, to an enzyme-product pair, EP, that was programmed to be an irreversible event. The cells designated as enzymes, E, were not permitted to move, and their random distribution in the grid limited them to a separation of at least 10 cells. Once a substrate molecule joined with an enzyme, no other pairings were possible. The lipophilicity of the substrate and product molecules were varied using the PB(WS) and PB(WP) rules. 

For competitive, reversible enzyme inhibition, the lowest measurable IC50 value is half of the enzyme concentration used in the assay (Cheng and Prusoff, 1973). From a practical view, kcJKM values of 104 M 1 s 1 and above are desirable for inhibitor profiling assays. With an enzyme-substrate pair characterized by a kcJKM value of 104 M 1 s, an assay can usually be run with a protease concentration in the single-digit nanomolar range in an automated setting. 

For some problems, such as the motion of heavy particles in aqueous solvent (e.g., conformational transitions of exposed amino acid sidechains, the diffusional encounter of an enzyme-substrate pair), either inertial effects are unimportant or specific details of the dynamics are not of interest e.g., the solvent damping is so large that inertial memory is lost in a very short time. The relevant approximate equation of motion that is applicable to these cases is called the Brownian equation of motion,... 

The lock-and-key model postulates that the structures of the enzyme and its substrate are complementary. In this way, the active site of the enzyme and the portion of the substrate to be acted on can fit closely together. The structures of these portions of the molecules are unique to the particular enzyme-substrate pair. They fit together like a particular key is necessary to open a specific lock. 

ELISAs (enzyme-linked immunoabsorbent assays) are another common framework used for drug screening. An enzyme-linked-antibody takes the place of a ligand, whose receptor is bound to a plate or filter. The mixture of drug and enzyme-linked antibody is incubated in the well with the receptor. After a series of washes to remove unbound material, the substrate for the enzyme is added to the well. A common enzyme/substrate pair is alkaline phosphatase and pNPP ( -nitrophenyl phosphate), which results in a yellow color. Another... 

There are also possibilities to detect other species with the gas sensitive field effect structures. Palladium gate devices are, e.g., sensitive to H2, H2S and at elevated temperatures also to, e.g., alcohols. Although IrTMOS structures, when operated up to 200°C, are highly selective to NH3 (only H2, low molecular amines and water vapor will interfere), it has been shown that PtTMOS structures operated at temperatures above 170°C are sensitive to a various degree also to alcohols, unsaturated hydrocarbones, ketones, and also some other organic compounds.It is therefore possible to use the ideas presented in this paper also for other than ammonia producing enzyme-substrate pairs. 

Enzymes are protein molecules which catalyse chemical transformations of specific molecules (substrates). An integral step in the catalytic mechanism is the binding of the substrate to the enzyme (see section 1.2.3). Thus enzymes combine molecular recognition with molecular transformation. Amongst the nearly 2000 known enzymes there is a wide range of binding specificity and some representative examples of enzyme-substrate pairs are shown in Table 1.3. An advantage of enzymes in the context of biosensors is that... 

First, some enzymes have broad specificity towards a wide range of substrates. These enzymes are potential workhorses and can be used for many polymer reactions. They are also good starting enzymes to use to screen for new reactions. A second case involves enzymes that are specific towards their own substrates but may still have some reactivity, albeit limited, towards other materials. In this case, the product yield may vary widely, depending on individual cases. Similarly, the reaction rate may vary depending on the enzyme-substrate pair, solvent, and other reaction conditions. In the (infrequent) happenstance, an enzyme may be found to have fortuitously good reactivity towards a non-substrate polymer under some specific reaction conditions. 

Figure 14 Schematic of the experimental setup for the rotating disk electrode (RDE) detection in a bead-based immunoassay using the ALP-PAPP enzyme-substrate pair.

Enzymes occur naturally Enzymatic reactions are specific The chemistry to incorporate enzyme-sensitive functional groups is simple and readily available Typical enzyme-sensitive groups (DNA, peptides) are very versatile and can be easily tuned to match an enzyme using the same chemical procedures A large number of enzyme/substrate pairs are available Responsiveness of one material to more than one enzyme is possible Transitions are reversible... 

It is a common understanding that the spatial arrangements of the substituents of a molecule have an crucial effect on whether an enzyme can accept the compound as a substrate. The effect of configuration on the difference of reactivities of enantiomers may be evaluated, as the two enantiomers can be separated and treated as individual starting materials and their products. In fact, promising models of enzyme-substrate interactions have been proposed that permit successful interpretation of the difference of reactivities between a given pair of enantiomers [29,30]. On the other hand, analysis of the reactivity of the conformational isomers of a substrate is rather difficult,because conformers are readily interconvertible under ordinary enzymatic reaction conditions. 

For this reason, these alternative routes for isotope combination with enzyme-substrate and/or enzyme-product complexes ensures that raising the [A]/[Q] or [B]/[P] pair will not depress either the A< Q or the B< P exchanges. Fromm, Silverstein, and Boyer conducted a thorough analysis of the equilibrium exchange kinetic behavior of yeast hexokinase, and the data shown in Fig. 2 indicate that there is a random mechanism of substrate addition and product release. 

Enzyme preparations of three Pieris species each contained B-thioglucosidase activity, a fact previously reported (36), and each inhibited hydrolysis in one or more combinations with a plant enzyme-substrate system. These data are being quantified and extended to include insect/preferred host-plant pairs. 

Complementary structures of biological materials, especially those of proteins, often result in specific recognitions and various types of biological affinity. These include many pairs of substances, such as enzyme-inhibitor, enzyme-substrate (analog), enzyme-coenzyme, hormone-receptor, and antigen-antibody, as summarized in Table 11.2. Thus, bioaffinity represents a useful approach to separating specific biological materials. 

M Meldal, K Breddam. Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal Biochem 195 141-147, 1991. 

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