Cyclic Enzyme Reactions

The basic principle of cycling sensors is the use of an enzyme pair which continuously cycles the substrate, S, and therefore causes a relatively large concentration change of an electrode-active cosubstrate, H (Scheller et al., 1985b Schubert et al., 1985a, 1986b Mizutani et al., 1985 Kulys et al., 1986a)  

An explicit solution of the differential equations requires the assumption 

This is only a minor restriction, since the recycled substrates are usually similar. The following formulas are obtained (Schulmeister, 1987b)  

The concentration profile typical for the diffusion of the substrate and the formation of the product up to a stationary level is shown in Fig. 36. 

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In enzyme electrodes, which are deliberately operated under conditions of diffusion control, the diffusion limits the sensitivity. Here, the coupling of cyclic enzyme reactions gives rise to a sensitivity enhancement by overcoming the limit set by diffusion. The excess of enzyme present in the membrane is included in the substrate conversion. On the other hand, the upper limit of linearity and the operational stability are decreased. 

ELIS A amplification techniques are sometimes used to generate 10-fold increases in absorbance values (55,64). An amplification system supplied by Gibco BRL (Gaithersburg, MD cat. no. 9589SA), uses alkaline phosphatase in the antibody conjugate to generate NADH from NADPH (Figure 3). The NADH is used as substrate in a secondary cyclic enzyme reaction in which a tetrazolium salt is reduced to a formazan dye, an intensely colored product that is measured spectrophotometrically. 

Enzyme Assay. Na , K -ATPase, and sarcoplasmic reticulum Ca - ATPase were prepared from rat hearts (22) and dog hearts (23), respectively. Bovine heart cyclic AMP phosphodiesterase was purchased from Sigma. The enzyme reaction was carried out after 5-min pretreatment with the drug, and the amount of inorganic phosphate liberated during the reaction period was determined. 

Models for biochemical switches, logic gates, and information-processing devices that are also based on enzymic reactions but do not use the cyclic enzyme system were also introduced [76,115,117-122]. Examples of these models are presented in Table 1.3. It should also be mentioned that in other studies [108,112-114,116], models of chemical neurons and chemical neural networks based on nonenzymic chemical reactions were also introduced. 

Coupled cyclic enzyme system Ii and I2 are inputs to the system s pools of substrate Xi and X3, respectively simple mass action kinetics irreversible reactions. 

This study is also based on the cyclic enzyme system, butits leading concept is to accomplish practical implementation of this system using biomaterials. In this respect, the analytical models developed here are related to several biochemical reactors in which enzymic reactions take place. This practical approach cannot be found in the models reviewed [76-86,109-122]. 

To examine the cyclic enzyme system proposed by Okamoto et al. [76-86] as an information-processing unit when this model is implemented in an experimental system and the enzymic reactions take place... 

Biochemically, folacin functions in vivo as coenzymes and carriers of one-carbon units for a number of enzyme reactions, including synthesis of amino acids, proteins, and nucleic acids (58,120,122). Folacin participates in both anabolic and catabolic reactions, and its metabolism is cyclic in nature. Greater detail on the biochemistry of folacin is available (120,122). 

The above discussion emphasized phosphorinane ring orientation in isolated and structurally characterized cyclic oxyphosphoranes and their relation to proposed P activated states in enzyme reactions of cAMP. Here, we concentrate on ring conformation and its projected role in cAMP interactions based largely on our recent structural work and preliminary investigations of new systems. 

By retro synthetic analysis collagenase inhibitor RO0319790 (1) can be assembled from two chiral building blocks, (R) -succinate 2 and (S)-tert-leucine N-methyla-mide 13. As the latter can be prepared from commercially available (S)-tert-leucine 8 our work concentrated in particular on the construction of the first building block 2. In order to assemble the carbon skeleton of 2 in the most efficient way, extremely cheap maleic anhydride 4 was converted in a known ene reaction with isobutylene to provide the cyclic anhydride 6. Hydrogenation of the double bond followed by the addition of EtOH/p-TsOH yielded the racemic diethyl ester substrate 9 for the enzyme reaction. The enzymatic monohydrolysis of 9 afforded the monoacid (R)-2a. (R)-2 a was coupled via its acid chloride with leucine amide 13 to ester 14, which finally was converted into the hydroxamic acid 1. 

Classically, the bell-shaped dependence of rate of the enzymic reaction on pH has been attributed to general acid and base catalysis by the two histidine residues in the active site, His-12 and His-119 (66). Support for this explanation based on the kinetic properties of a model system was first provided by an observation by Breslow and co-workers that 8-cyclodextrin functionalized with two imidazole groups will catalyze the 1,2-cyclic phosphate of 4-rert-butylcatechol (67). The dependence of hydrolysis rate on pH mimics that of RNase A, and this behavior demonstrates that the presence of two imidazole functional groups on a nonionizable framework is the simplest kinetic mimic of the enzyme. 

If a compound is nonfluorescent, it may be converted to a fluorescent derivative. For example, nonfluorescent steroids may be converted to fluorescent compounds by dehydration with concentrated sulfuric acid. These cyclic alcohols are converted to phenols. Similarly, dibasic acids, such as malic acid, may be reacted with j8-naphthol in concentrated sulfuric acid to form a fluorescing derivative. White and Argauer have developed fluorometric methods for many metals by forming chelates with organic compounds (see Ref. 23). Antibodies may be made to fluoresce by condensing them with fluorescein isocyanate, which reacts with the free amino groups of the proteins. NADH, the reduced form of nicotinamide adenine dmucleotide, fluoresces. It is a product or reactant (cofactor) in many enzyme reactions (see Chapter 24), and its fluorescence serves as the basis of the sensitive assay of enzymes and their substrates. Most amino acids do not fluoresce, but fluorescent derivatives are formed by reaction with dansyl chloride. 

In many of the examples cited of affinity labeling of enzymes by periodate-oxidized nucleotides, it has been assumed that the reaction involved formation of a Schiff base with an enzymic lysine, as in Fig. 3a,b. However, in very few papers has direct evidence been presented supporting the existence of a Schiff base intermediate. Lowe and Beechey (83), after examining in detail the structure of periodate-oxidized ATP, concluded that in aqueous solution there is little free aldehyde rather, the compound exists predominantly as an equilibrium mixture of three dialdehyde monohydrates (cyclic hemiacetals) and a dihydrate. The presence of cyclic hemiacetals may account for the ability of periodate-oxidized NADP and NADPH to function as coenzymes in several enzymic reactions (e.g., 78, 81). In many cases, the product of the covalent reaction of an enzyme and periodate-oxidized nucleotide may be a dihydroxymorpholino derivative (Fig. 3c), which is similar to the cyclic hemiacetals observed in aqueous solu-... 

Yokoyama, K. Kayanuma, Y. Cyclic voltammetric simulation for electrochemically mediated enzyme reaction and determination of enzyme kinetic constants. Anal Chem. 1998, 10, 3368—3376. 

Recently new methods, based on petturbations on the linear sweep voltammetry response of the mediator in the presence of the protein," a mediated thin-layer voltammetry technique, cyclic voltammetric simulation apphed to an electrochemically mediated enzyme reaction" have been setded to gain information on the protein-mediator interactions. More recendy the Scanning Electrochemical Microscopy (SECM) was used to probe the red-ox activity of individual cells of purple bacteria, by using two groups of mediators (hydrophilic and hydrophobic species) in order to gain information on the dependence of measured rate constant on the formal potential of the mediator in solution. By this technique an evaluation of the intracellular potential was also performed. ... 

As an example of an enzyme reaction which has been studied with fast-reaction techniques, we consider the mechanism of action of pancreatic ribonuclease A. Ribonuclease catalyzes the breakdown of ribonucleic acid in two distinct steps as shown in Fig. 9-6. First the diester linkage is broken, and a pyrimidine 2 3 -cyclic phosphate is formed then the cyclic phosphate is hydrolyzed to give the pyrimidine 3 -monophosphate and purine oligonucleotides with a terminal pyrimidine 3 -phosphate. Ribonuclease has been extensively studied with a variety of chemical and physical techniques, and its three-dimensional structure is known (cf. Richards and Wyckoff [12] for a comprehensive review). 

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