Enzymes sequential systems

The use of a catalyst with oxidase enzyme is an example of the use of a combined enzyme system, which illustrates the wide potential offered by multi-enzyme electrode systems. Various enzymes can be arranged to work sequentially to transform quite complex substances and eventually produce a measurable concentration-dependent change, which is detected by the output signal and recorded for analysis. 

Kapdan IK, Alparslan S (2005) Application of anaerobic-aerobic sequential system to real textile wastewater for color and COD removal. Enzyme Microb Technol 36 273-279... 

In a sequential reaction, all the substrates must be bound to the enzyme before any release of product can occur. Sequential systems can be either ordered or random. In an ordered sequential reaction, substrates must bind to the enzyme in a particular order, whereas in a random sequential system, substrates may bind to the enzyme in any order. In reaction schemes, substrates are usually abbreviated as A, B, C, and D in the order that they bind to the enzyme, whereas products are abbreviated as P, Q, R, and S in the order that they leave the enzyme. Sequential binding of substrates is a consequence of their orientation within the enzyme active site. 

It has also been demonstrated that expensive substrates such as UDP-Gal can be readily prepared in situ by enzymatic conversion of the relatively inexpensive sugar nucleotide uridine 5 -diphospho-a-D-glucopyranose (UDP-Glc) using a UDP-Gal 4-epimerase enzyme. This system, coupled with an appropriate UDP-Gal transferase, provides more economic access to enzymatically galactosylated compounds. In these multienzyme systems, to increase enzyme efficiency and also avoid multiple fermentations for separate enzyme preparations, fusion proteins have been constructed that contain both the Gal-epimerase and Gal-transferase enzymes. The use of these fused enzyme systems has increased in the recent years as their catalysis of sequential reactions can have a kinetic advantage over the mixture of two separated enzymes since the product of the first enzyme travels a shorter distance before being captured by the next enzyme in the sequence. 

There is no final consensus on whether procyanidin biosynthesis is controlled thermodynamically or enzymatically. In either case proanthocyanidins are synthesized through sequential addition of flavan-3,4-diol units (in their reactive forms as carbocations or quinone methides) to a flavan-3-ol monomer [218]. Based on the latest findings there is some evidence that different condensation enzymes might exist which are specific for each type of flavan-3,4-diol [64] and that polymer synthesis would be subject to a very complex regulatory mechanism [63]. But so far, no enzyme synthetase systems have been isolated and enzymatic conversion of flavanols to proanthocyanidins could not be demonstrated in vitro [219]. If biosynthesis was thermodynamically controlled, the variation in proanthocyanidin composition could be explained by synthesis at different times or in different compartments [64], The hypothesis of a thermodynamically controlled biosynthesis is based on the fact that naturally and chemically synthesized procyanidin dimers occur as a mixture of 4—>8 and 4—>6 linked isomers in approximate ratios of 3-4 1 [220]. Porter [164] found analogous ratios of 4—>8 and 4—>6 linkages in proanthocyanidin polymers. 

This complex synthesis was accomphshed following the definition of sequential cascade reactions combining multiple biocatalytic reactions without the necessity of intermediate work-up. The central intermediate of dTDP-activated deoxysugar synthesis was produced in an enzyme module system with sucrose synthase... 

NHase-AMase cascade system that naturally acts as a sequential system can be adequately decoupled by feeding the bioreactor with the appropriate substrate. The NHase activity was evaluated at various temperatures in the presence of acrylonitrile [20], propionitrile [18], benzonitrile [21], and 3-cyanopyridine [22] as substrates, while the AMase activity was separately investigated with the corresponding amides [23], The kinetics of both enzymes examined in an appropriate substrate concentration range were reported to follow the Michaehs-Menten equation, as reviewed previously [24],... 

FIGURE 18.5 Schematic representation of types of multienzyme systems carrying out a metabolic pathway (a) Physically separate, soluble enzymes with diffusing intermediates, (b) A multienzyme complex. Substrate enters the complex, becomes covalently bound and then sequentially modified by enzymes Ei to E5 before product is released. No intermediates are free to diffuse away, (c) A membrane-bound multienzyme system. 

Complement is not a single protein but comprises a group of functionally linked proteins that interact with each other to provide mar of the effector functions of humoral immunity and inflammation. Most of the components of the system are present in the serum as proenzymes, i.e. enzyme precursors. Activation of a complement molecule occurs as a result of proteolytic cleavage of the molecule, which in itself confers proteolytic activity on the molecule. Thus, many components of the system serve as the substrate of a prior component and, in turn, activate a subsequent component. This pattern of sequential activation results in the system being called the complement cascade. ... 

According to the preceding results we cannot determine the steady state of the system using the sequential approach suggested by Woodley [27]. This method involves sequential study of two phenomena reactant transfer in biphasic medium and enzyme kinetics in the aqueous medium. In the steady state, substrate transfer rate is equal to the reaction rate. 

Recently [63], we studied the behavior of two-enzyme system catalyzing two consecutive reactions in a macroheterogeneous medium (modified Lewis cell). The system consisted of lipase-catalyzed hydrolysis of trilinolein and subsequent lipoxygenation of liberated fatty acids (Fig. 3). Our approach compared the kinetic behavior of coupled enzymes in the Lewis cell with the sequential study of separated phenomena presented before ... 

Isik M, Sponza DT (2006) Biological treatment of acid dyeing wastewater using a sequential anaerobic/aerobic reactor system. Enzyme Microb Technol 38 887-892... 

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. 

Figure 2. A multistep enzyme interconversion cascade illustrating the sequential modification and conversion of target enzymes from one state of activity to another. Although written here as a cascade of increasing catalytic activity, enzyme-catalyzed covalent modification can either activate or inhibit target enzymes, depending on the particular system under study.

A sequential enzyme-catalyzed reaction scheme in which two substrates (A and B) react and form a single product and in which the substrates bind to the enzyme in a distinct order (i.e., only A and the product P can bind to the free enzyme). The reverse scheme of this mechanism is the ordered Uni Bi system. (See also Ordered Uni Bi Mechanism)... 

A sequential enzyme-catalyzed binding mechanism for a two substrate-two product system in which substrates A and B have to bind in a certain order but either P or Q can be released in a Theorell-Chance step upon the binding of B. Following this step, the other substrate is... 

Bioprocesses incorporating more than one redox enzyme in an oxidative reaction system might involve, in the simplest case, two oxidizing enzymes coupled so that they act sequentially to effect two oxidation steps. A key issue in the development of such oxidative biocatalytic systems would be the determination of the values, for each enzyme involved, of the redox potentials. These can be determined by potentiometric titration using redox mediators (such as NADH) and techniques such as cyclic voltammetry or electrophoresis [44]. Knowledge of the redox potentials would facilitate the design and engineering of a process in which the two... 

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