Multienzyme catalysis

Microreactors are ideal for directing complex enzymatic synthesis, such as multienzyme catalysis and cascade reactions. There have been a grovdng number of studies [173-178] that represent the implementation of microreactions for multistep enzymatic catalysis and a list is presented in Table 10.5. Microfluidic biocatalytic... 

Table 10.5 Immobilized enzyme microreactors for multienzyme catalysis.

In most multienzyme systems, the first enzyme of the sequence is a regulatory enzyme. This is an excellent place to regulate a pathway, because catalysis of even the first few reactions of a sequence that leads to an unneeded product diverts energy and metabolites from more important processes. Other enzymes in the sequence are usually present at levels that provide an excess of catalytic activity they can generally promote... 

The deprotonation and addition of a base to thiazolium salts are combined to produce an acyl carbanion equivalent (an active aldehyde) [363, 364], which is known to play an essential role in catalysis of the thiamine diphosphate (ThDP) coenzyme [365, 366]. The active aldehyde in ThDP dependent enzymes has the ability to mediate an efScient electron transfer to various physiological electron acceptors, such as lipoamide in pyruvate dehydrogenase multienzyme complex [367], flavin adenine dinucleotide (FAD) in pyruvate oxidase [368] and Fc4S4 cluster in pyruvate ferredoxin oxidoreductase [369]. 

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. 

A multienzyme complex can carry out the coordinated catalysis of a complex reaction. The intermediates in the reaction remain bound to the complex and are passed from one enzyme component to the next, which increases the overall reaction rate and minimizes side reactions. In the case of isolated enzymes, the reaction intermediates would have to diffuse randomly between enzymes. 

Preparation and characterization of liposomes formed with natural phospholipids were well established. However, in using liposomes for simulation of enzymatic functions, especially in acid-base catalysis, difficulties would be encountered due to their chemicai and morphological instabilities. Thus, bilayer membranes composed of synthetic amphiphiles are more favorable candidates for enzyme mimics. For example, artificial vitamin Bg-dependent enzymes were constructed from catalytic bilayer membranes in combination with a bilayer-forming peptide lipid (10), a hydrophobic vitamin derivative (11), and metal ions (Fig. 5). The catalyst acts as an artificial aminotransferase, showing marked substrate specificity, high enantioselectivity, and turnover behavior for the transamination of a-amino acid with a-keto acids. In addition, the reaction fields provided by the catalytic bilayer membranes are suitable to establish multienzyme systems through functional ahgnments of artificial enzymes and natural ones in a sequential manner. 

The accumulation of pyruvic acid in treated Aspergillus points to the molecular site of action of DMDC, oxine, and pyrithione, namely catalysis of the oxidative destruction of dihydrolipoic acid [thioctic acid 2.28) (Sijpesteijn and Janssen, 1959). This is the essential coenzyme for oxidative decarboxylation of pyruvic acid by dihydrolipoylacetyltransferase, a component of the multienzyme complex known as pyruvate dehydrogenase. 

Since the beginning of enzyme catalysis in microemulsions in the late 1970s, several biocatalytic transformations of various hydrophilic and hydrophobic substrates have been demonstrated. Examples include reverse hydrolytic reactions such as peptide synthesis [44], synthesis of esters through esterification and transesterification reactions [42,45-48], resolution of racemic amino acids [49], oxidation and reduction of steroids and terpenes [50,51], electron-transfer reactions, [52], production of hydrogen [53], and synthesis of phenolic and aromatic amine polymers [54]. Isolated enzymes including various hydrolytic enzymes (proteases, lipases, esterases, glucosidases), oxidoreductases, as well as multienzyme systems [52], were anployed. 

The HRP-catalyzed polymerization of phenols was found to be a convenient way to produce redox polymers and conducting (electronically conducting and ionically conducting) polymers. Besides the interest in electronic conductive polyanilines [121], many efforts have been made to produce ionically conductive phenol polymers for battery applications. A classic effort is the synthesis of poly(hydroquinone) for use as a redox polymer. Typically, poly(quinone)s are prepared via chemical or electrochemical methodologies [122,123]. Both processes produce a large amount of by-products and lead to complex polymer structiues. The first alternative pathway to produce poly(hydroquinone) by peroxidase catalysis was based on a multienzymic... 

These biochemical transformations occur on a multienzyme complex composed of at least three dissimilar proteins biotin carrier protein (MW = 22,000), biotin carboxylase (MW = 100,000) and biotin transferase (MW = 90,000). Each partial reaction is specifically catalyzed at a separate subsite and the biotin is covalently attached to the carrier protein through an amide linkage to a lysyl a-amino group of the carrier protein (338, 339). In 1971, J. Moss and M. D. Lane, from Johns Hopkins University proposed a model for acetyl-CoA carboxylase of E, coli where the essential role of biotin in catalysis is to transfer the fixed CO2, or carboxyl, back and forth between two subsites. Consequently, reactions catalyzed by a biotin-dependent carboxylase proceed though a carboxylated enzyme complex intermediate in which the covalently bound biotinyl prosthetic poup acts as a mobile carboxyl carrier between remote catalytic sites (Fig. 7.13). 

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