Enzymes coupled systems

End-point assays can also use coupled enzyme systems in which the product of the test reaction provides the substrate for the subsequent auxiliary and indicator reactions. The choice of pH is less critical than with the measurement of enzyme activity frequently a compromise pH is used and... 

The system can be applied for examination of control mechanisms of metabolic coupled enzyme systems, such as the sugar transport system in bacteria. 

Yang and Schulz also formulated a treatment of coupled enzyme reaction kinetics that does not assume an irreversible first reaction. The validity of their theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A as a substrate. Unlike the conventional theory, their approach was found to be indispensible for coupled enzyme systems characterized by a first reaction with a small equilibrium constant and/or wherein the coupling enzyme concentration is higher than that of the intermediate. Equations based on their theory can allow one to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre-steady state. 

Another approach of using fluorimetric assays for screening purposes is the use of coupled enzyme systems. McElroy et al. presented an assay for glutamate... 

Scheme 10. The synthesis routes to F-labelled nucleosides and ribose in coupled enzyme systems [20].

With over-expressed fluorinase and under optimised reaction conditions, a synthesis of [ F]-FDA 5d from [ F]-fluoride was achieved in a radiochemical yield (RCY) of 95%. Also in coupled enzyme systems, where the fluorinase is coincubated with other enzymes, the syntheses of [ F]-5 -fluoro-5 -deoxy-inosine... 

I am disturbed that following Professor Hammes presentation the language in the discussion has changed. While discussing enzymes we looked at molecule properties closely related to the discussion of small molecules (e.g., atom position and motions). Now in the discussion of complex enzymes, especially in membranes, we have started to use bulk properties (e.g., we talk of phases, dielectric constants, Chapman-Gouy theory, etc.). Is it the view of the discussants that events in membrane-coupled-enzyme systems cannot be described by molecular events because of the complexity of the system resulting from extensive cooperativ-ity within the membrane (e.g., between lipids and proteins) ... 

A nonlinear relationship between enzyme concentration and measured activity is indicative of a more complex reaction system. Complications of this nature may arise from such things as changes in the composition of the reaction mixture (e.g., pH due to the addition of increasing amounts of enzyme solution), assay limitations (e.g., insufficient substrate), limited coupling-enzyme (where assays are based on coupled enzyme systems), the presence of inhibitors, and enzyme-cofactor or enzyme-enzyme dissociation phenomena. Nonlinear relationships may also be an inherent... 

Coupled enzyme systems have been used in enzymology to significantly increase the sensitivity of detection of enzyme-substrate reactions. The addition of coupled enzyme systems to an enzyme immunoassay should lead to several orders of magnitude increase in the sensitivity (48, 67). 

Because of the lack of good experimental data in human brain chemistry, our presentation is limited to the use of carefully chosen normalized experimental parameters in order to reproduce the basic static and dynamic characteristics of this coupled enzymes system. 

Akyilmaz E, Sezgintiirk MK, Dinfkaya E (2003) A biosensor based on urate oxidase-peroxidase coupled enzyme system for uric acid determination in urine. Talanta 61 73-79... 

This coupled enzyme system is used by the blue-green algae and by Rhizobia. 

Several organic solvents were investigated with regard to stability and activity of HLADH as well as their influence on the hydrogenase-driven reaction. Hydrophobic solvents such as heptane or toluene proved to be the most suitable solvents for the coupled enzyme-system. Furthermore, it became apparent that nonimmobilized cells, permeabilized with cetyl-trimethylammonium bromide, showed the best results for NADH regeneration. After optimization the conversion in heptane with 10% water yields 99% cyclohexanol by reduction of cyclohexanone. 

Figure 9.73 Kinetics of the coupled enzyme system a-amylase-a-glucosidase, with maltoheptaose in mole percent. (From Haegele et al., 1981.)...

The source of the label may be the solvent or a coupled substrate. It can be introduced by a single enzyme (Scheme 57) or using a coupled enzyme system (Scheme 58). ... 

A cardiologist is studying the effect of alcohol on triglyceride accumulation in rat heart. The triglycerides are saponified and the glycerol released is determined by the coupled enzyme system shown below. 

When making initial rate measurements with coupled enzyme systems, there is often a significant lag time during which the linkage products build up to steady-state concentrations.6 Remember that some dehydrogenase reactions possess unfavorable equilibria lactate dehydrogenase, for example, which catalyzes the reaction shown in Eq. 3.12 ... 

Before measurements one vial of enzymes was diluted by 0.5 mL of 0.1 mol/L phosphate buffer. For the coupled enzyme system the reaction mixture contained 10 /iL Lu+R, 50 juL 0.002 % tetradecanal, 200 juL 0.1 M phosphate buffer (pH 6.8), 200 pL 0.4 mM NADH and 50 pL 0.5 mM FMN. The cuvette was placed into the bioluminometer BLM 8801 (SKTB Nauka , Krasnoyarsk, Russia) and control light emission (L) was recorded. When the light emission reached a steady state 50 pL of a test water was pipetted into cuvette, and the light intensity (lex) was measured again. 

Before measurements one vial of bacteria was diluted by 500 pL 1.5 % NaCl. 20 pL of bacteria solution was added to 1 mL of 3 % NaCl and the control light intensity was recorded using the bioluminometer after 15 min period of incubation. The measurements were repeated with 1 mL 3 % NaCl prepared on water samples and experimental light intensity was measured. The effect of sample water on coupled enzyme system bioluminescence was estimated by the bacterial (BI) or luciferase (LI) index using the following formula BI = If lao LI = If I. ... 

Bioluminescence bioassays based on luminous bacteria and coupled enzyme system NADH-FMN-oxidoreductase-luciferase were adapted for monitoring the saline-water conditions of Lake Shira (Khakasia, Siberia). The differences in bioluminescence responses have been found to be related to the salt composition and the oxidation-reduction properties of water. Bioluminescent kinetics parameters, which are mostly sensitive to pollution under conditions of saline water, have been observed. Figure 1 shows the typical bioluminescence kinetics of the samples of water due to anthropogenic influence (beach) and control clear water (non-recreational area). 

The two enzymatic reactions are coupled by PEG-NADH and PEG-NAD+. This stoichiometric coupling does not affect enzyme kinetics but has to be considered when writing the mass balances. The discussion of this system will be continued in Sect. 7.5 where some implications of coupled enzyme systems on reactor design are described. 

Considerations about process optimization of coupled systems with coenzyme regeneration are discussed in the literature(29, 42,43, 13S. One aspect may be illustrated here - the question of enzyme ratio within the coupled enzyme system. 

As proof of the kinetic model, fitting of initial rate data or time-course data of batch reactions have been introduced in Sect. 7.4. Additionally, a proper fit of continuous reactions in an enzyme membrane reactor (EMR) may serve as confirmation of the kinetic model. For this coupled enzyme system, calculated and measured conversions at different operating conditions (varying [E] and t values, not further specified) are presented in Fig. 7-34. 

J. D. Rozzell, Production of Amino Acids l/s-ing Coupled Enzyme Systems, 1989, U. S. Patent 4,880,738. 

Leucine dehydrogenase is the enzyme used as the biocatalyst in the process commercialized by Degussa to produce L-tert-leucine (9). Similar to phenylalanine dH, leucine dH has been used to prepare numerous unnatural amino acids because of its broad substrate specificity. For example, cloned, thermostable alanine dH has been used with a coupling enzyme system to prepare D-amino acids. - ... 

This coupled enzyme system is used by blue-green algae and by Rhizobium. The amide group of glutamine provides the ammonia for the synthesis of many //-containing compounds, e.g., purines and pyrimidines. 

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