Multiple enzyme and substrate

Inhibition of Enzyme Reactions 391 Multiple Enzyme and Substrate Systems 392... 

Our discussion of enzyme.s is continued in the Professional Reference Shelf on the DVD-ROM and on the Web where we describe multiple enzyme and. substrate systems, enzyme regeneration, and enzyme co-tactors (see R9.6). 

A titrametric assay of PLCSc, alternatively called the pH-stat method, was the workhorse in early studies [28]. This method simply involves titrating the acidic product of the PLC reaction as it is formed with a solution of standard base. An advantage of this continuous assay is that it can be used to detect the turnover of both synthetic and natural substrates, and its sensitivity has been estimated to be in the 20-100 nmol range. However, the pH-stat assay has low throughput capability, and it cannot be easily performed in a parallel fashion with multiple substrate concentrations. It is also necessary to exclude atmospheric carbon dioxide from the aqueous media containing the enzyme and substrate. 

Most, if not all, milks contain sufficient amounts of lipase to cause rancidity. However, in practice, lipolysis does not occur in milk because the substrate (triglycerides) and enzymes are well partitioned and a multiplicity of factors affect enzyme activity. Unlike most enzymatic reactions, lipolysis takes place at an oil-water interface. This rather unique situation gives rise to variables not ordinarily encountered in enzyme reactions. Factors such as the amount of surface area available, the permeability of the emulsion, the type of glyceride employed, the physical state of the substrate (complete solid, complete liquid, or liquid-solid), and the degree of agitation of the reaction medium must be taken into account for the results to be meaningful. Other variables common to all enzymatic reactions—such as pH, temperature, the presence of inhibitors and activators, the concentration of the enzyme and substrate, light, and the duration of the incubation period—will affect the activity and the subsequent interpretation of the results. 

The decisive experiment of Biebricher and Luce is shown in Fig. 4. A synthesis medium containing highly purified enzyme and substrates is incubated and maintained at a suitable temperature, for a time adequate to allow the multiplication of any templates present but too short to enable products to arise de novo. Then the solution is divided into portions. Each portion is incubated long enough to allow synthesis de novo and the products are compared by the fingerprint method. If the impurity hypothesis is correct, then multiplication of the impurity in the first phase should lead to the same product from each portion of the incubated medium. If the de novo hypothesis is correct then the products should be different, since at the beginning different enzyme molecules were working on different products. Selection, that is, preferential reproduction of one rudimentary strand, could not yet take place, since in the first, short incubation none of the products de novo was complete. 

In retrospect, Cushny was one of the earliest investigators to point out the diastereoisomeric situations which occur when a chiral biopolymer interacts with the enantiomeric forms of another molecule. The three-point attachment and polyaffinity concepts provided an easily visualized picture of how the differentiation might occur. For a time, unfortunately, the possibility of differentiation by a one-point approach was not clearly recognized and three-point attachment became somewhat dogmatic. With more detailed structural investigations, it is becoming clear that interactions between enzymes and substrates, and receptors and drugs, often involve a multiplicity of interactions. 

Enzyme reactions proceed, in general, via several intermediate states. A simple model incorporating multiple states is shown below enzyme and substrate assodate to form an enzyme-substrate complex, which undergoes a conformational change to ES before breaking down into enzyme and product... 

In the preceding section we discussed how the addition of a second substrate, I, to enzyme-catalyzed reactions could deactivate the enzyme and greatly inhibit the reaction. In the present section we look not only at systems in which the addition of a second substrate is necessary to activate the enzyme, but also other multiple-enzyme and multiple-substrate systems in which cyclic regeneration of the activated enzyme occurs. 

We model the hinge-bending motion associated with interactions between the enzyme and substrate as a classical particle one-dimensional multiple-step random walk in the presence of a force field [12,24], n(f), where n t) is the step index (Fig. 24.7). The position distribution density function Pn t) can be calculated [12] by... 

A rigorous kinetic description of interfacial catalysis has been hampered by the ill-defined physical chemistry of the lipid—water interface (Martinek et ai, 1989). Traditional kinetic assumptions are undermined by the anisotropy and inhomogeneity of the substrate aggregate. For example, the differential partitioning of reactants (enzyme, calcium ion, substrate) and products (lysolecithins, fatty acids) between the two bulk phases prevents direct measurement of enzyme and substrate concentrations. This complicates dissection of the multiple equilibria that contribute to the observed rate constants. Only recently has it become possible to describe clearly the activity of SPLA2S in terms of traditional Michaelis— Menten kinetics. Such a description required the development of methods to reduce experimentally the number of equilibrium states available to the enzyme (Berg etai, 1991). 

Miniaturized proteomic analysis devices include enzyme assays and immunoassays. The enzyme assay chip is mainly a sophisticated incubator and flowthrough system. It can perform multiple functions typically required by the biochemist, namely, diluting substrate and buffer, mixing enzyme and substrate, incubating during conversion, and allowing for detection in a flow channel. Immunoassay chips are similar... 

The mechanism by which multiple attack takes place is not at all clear, although the finding that pig pancreatic amylase has two apparently independent binding sites for substrates and forms multimolecular enzyme-substrate complexes (127) suggests a way in which enzyme and substrate could be constrained to continue the association necessary for multiple attack. 

Although there is no disagreement concerning the appearance of the endoplasmic reticulum on thin section, the tridimensional reconstruction of this planar structure is debated. The controversy is easier to understand if the proposed roles of the endoplasmic reticulum in biology are kept in mind. Among these hypothetical roles are (1) the endoplasmic reticulum provides compartments within the cytoplasm (2) it separates multiple-enzyme systems or separates the enzyme from its substrate (3) it provides a support for enzymes and substrates, thereby facilitating reaction and (4) it furnishes an intracellular circulatory system in connection with the exterior, which promotes rapid diffu-... 

We have demonstrated a concept of enzymatic biosensor by using electrochemical detection. The process generally involved two mechanisms of (1) chemical reaction and (2) electrochemical reaction. The initial chemical reaction, resulted from enzyme and substrate, is often found by interacting with multiple enzymes as shown by the example of cholesterol esterase and... 

For the purpose of the present discussion the term transient kinetics is applied to the time course of a reaction from the moment when enzyme and substrate are mixed, t=0, until either a steady state or equilibrium is established. The difference between the kinetic problems discussed in section 3.3 and in the present section is, respectively, the presence of catalytic as distinct from catalytic concentrations of enzyme. Here we are concerned with the stoichiometry of enzyme states. Transient kinetic experiments with enzymes can be divided into two types. The first of these (multiple turnover) is carried out under the condition that the initial concentrations of substrate and enzyme are Cs(0) Ce(0) and c it) can, therefore, be regarded as constant throughout the course of the reaction until a steady state is attained. Alternatively, in a single turnover reaction, when Cs(0)reaction intermediates is observed until the overall process is essentially complete. These two possibilities will be illustrated with specific examples. In connection with a discussion of the approach to the steady state, in section 3.3 it was emphasized that, at t = 0, the concentrations of the intermediates, enzyme-substrate and enzyme-product complexes, are zero and, therefore, the rate of product formation is also zero. Under the experimental conditions used for steady state rate measurements and for enzyme assays, the first few seconds after the initiation of a reaction are ignored. However, when the experimental techniques and interpretation discussed below are used, events during the first few milliseconds of a reaction can be analysed and provide important information. With suitable monitors it is possible to follow the formation and decay of enzyme complexes with substrates and... 

Such CEEAs can also be produced that contain two or more different enzymes in the same aggregates. These so-called combi-CLEAs allow multiple reactions (either cascade or noncascade) to be carried out in the same reaction space (Figure 4.2). This preparation strategy also has some advantages in promoting high specific-enzyme activity and catalytic productivity since reaction rates can be improved by close spatial proximity between the different enzymes and substrates. 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

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