Sequential coupling, enzyme reactions

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 ... 

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. 

Organisms Differ in Sources of Energy, Reducing Power, and Starting Materials for Biosynthesis Reactions Are Organized into Sequences or Pathways Sequentially Related Enzymes Are Frequently Clustered Pathways Show Functional Coupling The ATP-ADP System Mediates Conversions in Both Directions... 

As early as 1970, Clark patented the sequential coupling of two enzymes, e.g., for the determination of disaccharides. Since this type of reaction is widespread in metabolism it can also be carried out by using cell organelles or microorganisms. 

Another type of sequential coupling is provided by cycling reactions. The product of the primary enzyme reaction is regenerated to the substrate of this reaction, i.e., the analyte, in a second, enzyme-catalyzed reaction. These cycles are based on the dependence of the two enzymes on different cofactors thus, the required free enthalpy exists for both reactions. The analyte molecule may be regarded as a catalyst of the reaction between the two cofactors. This results in a rate of cofactor conversion and enthalpy production that is enormously higher than that in a single enzyme reaction. These cycling reactions therefore lead to a substantial increase of sensitivity. 

Figure 14-26. Basic principles of the coupling of enzyme reactions in biosensors, a) Sequential coupling ...

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

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... 

Three oxidations states are potentially available in a binuclear iron center. Enzymes with octahedral fi-o o bridged iron clusters can be isolated in each of the three states the diferric and diferrous states appear to be the functional terminal oxidation states for most of the enzymes, while the mixed valence state may be an important intermediate or transition state for some reactions (Que and True, 1991). In these enzymes the cluster participates primarily as a two-electron partner in the redox of substrates, perhaps using sequential one-electron steps. Without additional coupled redox steps the enzyme is in a new oxidation state after one turnover. In contrast only the diferric and mixed valence oxidation states have been found for 2Fe 2S clusters. The diferrous state may not be obtainable because of the high negative charge on [2Fe 2S(4RS)] versus -1 or 0 net charge for the diferrous octahedral (i.e., non-Fe S) clusters. The 2Fe 2S proteins either are one-electron donor/acceptors or serve as transient electron transfer intermediates. 

The animal fatty acid synthase (FAS EC 2.3.1.85) is one of the most complex multifunctional enzymes that have been characterized, as this single polypeptide contains all the catalytic components required for a series of 37 sequential transactions (Smith, 1994). The animal FAS consists of two identical polypeptides of approximately 2500 amino acid residues (MW, ca. 270 kDa), each containing seven catalytic subunits (1) ketoacylsynthase, (2) malonyl/acetyl transferase, (3) dehydrase, (4) enoyl reductase, (5) (3-kcto reductase, (6) acyl carrier protein (ACP), and (7) thioesterase. Although some components of the complex are able to carry out their respective catalytic steps in the monomeric form, only in the FAS dimer do the subunits attain conformations that facilitate coupling of the individual reactions of fatty acid synthesis to occur (Smith et al., 2003). 

In yeast and mycelial fungi, xylose is metabolized via coupled oxidation-reduction reactions . Xylose reductase is the enzyme involved in the reduction of xylose to xylitol. Sequential enzymatic events, through the oxidation of xylitol to xylulose, lead to the utilization of xylose. Many yeast species utilize xylose readily, but the ethanol production capability is very limited. Only a few yeast species effectively produce ethanol from xylose these include Pachysolen tan-nophilus, Candida shihatae and Pichia stipitis [80]. The production of ethanol from xylose by these three yeast strains has been studied extensively in recent years. Recently, genetically engineered yeast strains have been constructed for more effective conversion of xylose to ethanol. 

---