Sequential enzyme reactions applications

Easterby proposed a generalized theory of the transition time for sequential enzyme reactions where the steady-state production of product is preceded by a lag period or transition time during which the intermediates of the sequence are accumulating. He found that if a steady state is eventually reached, the magnitude of this lag may be calculated, even when the differentiation equations describing the process have no analytical solution. The calculation may be made for simple systems in which the enzymes obey Michaehs-Menten kinetics or for more complex pathways in which intermediates act as modifiers of the enzymes. The transition time associated with each intermediate in the sequence is given by the ratio of the appropriate steady-state intermediate concentration to the steady-state flux. The theory is also applicable to the transition between steady states produced by flux changes. Apphcation of the theory to coupled enzyme assays makes it possible to define the minimum requirements for successful operation of a coupled assay. The theory can be extended to deal with sequences in which the enzyme concentration exceeds substrate concentration. 

The application of immobilized model systems for investigation of the kinetics of sequential enzyme reactions has been reviewed. 

In DERA reactions, where acetaldehyde is the donor, products are also themselves aldehydes. In certain cases a second aldol reaction will proceed until a product has been formed that can cyclize to a stable hemiacetal.71 For example, when a-substituted aldehydes were used, containing functionality that could not cyclize to a hemiacetal after the first aldol reaction, these products reacted with a second molecule of acetaldehyde to form 2,4-dideoxyhexoses, which then cyclized to a hemiacetal, preventing further reaction. Oxidation of these materials to the corresponding lactone, provided a rapid entry to the mevinic acids and compactins (Scheme 5.43). Similar sequential aldol reactions have been studied, where two enzyme systems have been employed72 (Scheme 5.44). The synthesis of 5-deoxy ketoses with three substitutents in the axial position was accomplished by the application of DERA and RAMA in one-pot (Scheme 5.44). The long reaction time required for the formation of these thermodynamically less stable products, results in some breakdown of the normally observed stereoselectivity of the DERA and FDP aldolases. In a two-pot procedure, DERA and NeuAc aldolase have... 

An interesting, as yet unstudied, potential application of immobilized enzyme or catalytic membranes is their use in the conduct of sequential catalytic reactions, as illustrated in Fig. 9.1. Rapid and slow catalytic reactions with different catalysts can be conducted consequently in different converters fluid leaving one membrane converter can be delivered to a second catalytic membrane containing a different catalyst operative on the product of the first transformation. Since there can be no back diffusion from one membrane to the other, there is no chance for products of the second reaction to be acted upon by the first catalyst or to interact with the initial substrate. Thus, cross-reaction between different intermediates and different catalysts is avoided. This may allow continuous, sequential catalytic reactions, impossible to perform concurrently, to be carried out efficiently and rapidly. 

This chapter reviewed some of our group s contributions to the development and application of QM/MM methods specifically as applied to enzymatic reactions, including the use of sequential MD/QM methods, the use of effective fragment potentials for reaction mechanisms, the development of the new QM/MM interface in Amber, as well as the implementation and optimization of the SCC-DFTB method in the Amber program. This last implementation allows the application of advanced MD and sampling techniques available in Amber to QM/MM problems, as exemplified by the potential and free energy surface surfaces for the reaction catalyzed by the Tripanosoma cruzi enzyme /ram-sialidasc shown here. 

The FIA system consisted of TMI modules (Tecniques Mesura Instrumentacio), a five-channel peristaltic pump, an eight-channel injection valve, an eight-channel distributed valve, and a colorimeter connected to an interface and to an IBM-PC microcomputer. A specific application software, Qcontroll , was used for data acquisition, with a continuous historical trend, and a scheduled control program for sequential analysis. In a previous scheme as in Fig. 1, free enzymes were used and the whole system was calibrated for a linear range of 0.05-1.0 g of ethanol/ L of standard ethanol solutions, and the product of the reaction was passed through the colorimeter. 

In principle, such processes should also be applicable to bifunctional aldehydes for a two-directional chain elongation in which two equivalents of DHAP nucleophiles would be added sequentially to both the acceptor carbonyls in a fashion that can be classified as a tandem reaction [66,67], without the need for isolation of any intermediates. Depending on the specificity of the enzyme used and on the number and position of hydroxyl functions in the starting material, the isomeric constitution, as well as the absolute and relative stereochemistry should be deliberately addressable. Thus, in a preparatively simple manner, such tandem aldolizations [68] should permit to rapidly construct larger carbohydrate molecules that would rival the carbohydrate core of tunica-mine and related nucleoside antibiotics in structural complexity. 

Scheme 14 shows a proposed application in which PC is transformed into phosphatidylsolketal (PSK). This compound is hydrolyzed by PLCpc forming the OP corresponding to the alcohol acceptor in the first enzymatic reaction. The result of the sequential use of the two enzymes, one in transesterification and the second in hydrolysis, corresponds to the net phosphorylation reaction. The reaction can be of very wide applicability if the hypothesis is verified that both... 

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