Multienzyme system

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. 

The high catalytic activity of enzymes has a number of sources. Every enzyme has a particular active site configured so as to secure intimate contact with the substrate molecule (a strictly defined mutual orientation in space, a coordination of the electronic states, etc.). This results in the formation of highly reactive substrate-enzyme complexes. The influence of tfie individual enzymes also rests on the fact that they act as electron shuttles between adjacent redox systems. In biological systems one often sees multienzyme systems for chains of consecutive steps. These systems are usually built into the membranes, which secures geometric proximity of any two neighboring active sites and transfer of the product of one step to the enzyme catalyzing the next step. 

A common characteristic of metabolic pathways is that the product of one enzyme in sequence is the substrate for the next enzyme and so forth. In vivo, biocatalysis takes place in compartmentalized cellular structure as highly organized particle and membrane systems. This allows control of enzyme-catalyzed reactions. Several multienzyme systems have been studied by many researchers. They consist essentially of membrane- [104] and matrix- [105,106] bound enzymes or coupled enzymes in low water media [107]. 

Enzyme Catalyzed. The enzyme aldolases are the most important catalysts for catalyzing carbon-carbon bond formations in nature.248 A multienzyme system has also been developed for forming C-C bonds.249 Recently, an antibody was developed by Schultz and co-workers that can catalyze the retro-aldol reaction and Henry-type reactions.250 These results demonstrate that antibodies can stabilize the aldol transition state but point to the need for improved strategies for enolate formation under aqueous conditions. 

The pentose phosphate cycle represents a multienzyme system in... 

Franke, D., Machajewski, T., Hsu, C.-C. and Wong, C.-H. (2003) One-pot synthesis of L-fructose using coupled multienzyme systems based on rhamnulose-1-phosphate aldolase. The Journal of Organic Chemistry, 68 (17), 6828-6831. 

This equation is formally analogous to Hill s empirical equation (Hill, 1910) which is employed to rationalize allosteric interactions in multienzyme systems. 

K. F. Gu and T. M. S. Chang, Conversion of ammonia or urea into L-leucine, L-valine and L-isoleucine using artificial cells containing an immobilized multienzyme system and dextran-NAD+, glucose dehydrogenase for co-factor recycling, A 4/0, 11(1), 24-28 (1988). 

A catalytic function or region of a multienzyme system that is associated with a domain that is separate and distinct from the domain(s) responsible for the other catalytic function(s). 

Clearly, many of these effects can be addressed through preliminary studies. Incubation of the enzyme at one temperature while assaying at a different temperature will address issues concerned with protein stabihty as well as effects of temperature on any couphng enzymes in the assay protocol. Nevertheless, temperature studies on each enzyme in a multienzyme system should be addressed individually at an early point in the investigation. Issues related to the effects on affinities can usually be minimized by assaying under saturating conditions. Care... 

This review covers the recent literature (2002-2007) in which either multienzyme systems or, alternatively, true chemoenzymatic processes (i.e. those in which an enzyme is combined with a chemical catalysts/reagent in a key step) are employed for the synthesis of chiral amino acids and amines. Not included are those papers in which the term chemoenzymatic refers to the fact that the substrate for the biotransformation has simply been prepared via chemical synthesis. 

W. II. Pitcher Design and Operation of Immobilized Enzyme Reactors. - S. A Barker Biotechnology of Immobilized Multienzyme Systems. - R. A Messing Carriers for Immobilized Biologically Active Systems. -P. Brodelius Industrial Applications of Immobilized Biocatalysts. - B. Solomon Starch Hydrolysis by Immobilized Enzymers. 

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... 

In some multienzyme systems, the regulatory enzyme is specifically inhibited by the end product of the pathway whenever the concentration of the end product exceeds the cell s requirements. When the regulatory enzyme reaction is slowed, all subsequent enzymes operate at reduced rates as their substrates are depleted. The rate... 

Light-driven electron transfer in plant chloroplasts during photosynthesis is accomplished by multienzyme systems in the thylakoid membrane. Our current picture of photosynthetic mechanisms is a composite, drawn from studies of plant chloroplasts and a variety of bacteria and algae. Determination of the molecular structures of bacterial photosynthetic complexes (by x-ray crystallography) has given us a much improved understanding of the molecular events in photosynthesis in general. 

The reducing equivalents temporarily stored in N AD(P)H are utilized in a number of ways, all of which lead to biosynthesis of essential molecules and/or oxidative degradation of metabolites. Examples range from the simple reduction of a substrate for biosynthetic purposes (e.g. the steroid reductase mediated hydrogenation of the isolated double bond of desmosterol to give cholesterol, Table 1) (71MI11000) to complex electron transport chains that are switched on by the transfer of the electrons of N AD(P)H to the next electron carrier of the chain. These multienzyme systems are used for a number of purposes (see below). 

Oxidative decarboxylations of a-keto acids are mediated by either enzymes having more than one cofactor or complex multienzyme systems utilizing a number of cofactors. For example, pyruvate oxidase uses TPP and FAD as coenzymes, the function of the latter being to oxidize the intermediate (41). Conversion of pyruvate to acetyl-CoA requires a multienzyme complex with the involvement of no less than five coenzymes, TPP, CoA, dihydrolipoate, FAD and NAD+ (74ACR40). 

Y. Ichikawa, J. L.-C. Liu, G.-J. Shen, and C.-H. Wong, A highly efficient multienzyme system for the one-step synthesis of sialyl trisaccharide In situ generation of sialic acid and TV-acetyllactosamine coupled with regeneration of UDP-glucose, UDP-galactose, and CMP-sialic acid, J. Am. Chem. Soc. 113 6300 (1991). 

Heme coenzymes, iron-sulfur clusters, flavin coenzymes, and nicotinamide coenzymes cooperate in multienzyme systems to catalyze the chemically remarkable hy-droxylations of hydrocarbons such as steroids (chapter 20). In these hydroxylation systems, the heme proteins constitute a family of proteins known as cytochrome P450, named for the wavelength corresponding to the most intense absorption band of the carbon monoxide-liganded heme, an inhib-... 

Heme coenzymes participate in a variety of electron-transfer reactions, including reactions of peroxides and 02. Iron-sulfur clusters, composed of Fe and S in equal numbers with cysteinyl side chains of proteins, mediate other electron-transfer processes, including the reduction of N2 to 2 NH3. Nicotinamide, flavin, and heme coenzymes act cooperatively with iron-sulfur proteins in multienzyme systems that catalyze hydroxylations of hydrocarbons and also in the transport of electrons from foodstuffs... 

The bacterium Thiobacillus versutus has been shown to require manganese for both growth and oxidation of thiosulfate. This organism uses a multienzyme system that includes two colorless proteins, A and B, and two c-type cytochromes to oxidize thiosulfate to sulfate. Protein A is a thiosulfate-binding protein protein... 

The results from the publications mentioned are of interest because they can help in the creation of effective catalytic systems containing porphyrins, which combine functions typical of multienzyme systems. The task in hand is the possible synthesis of bifunctional catalysts based on metalloporphyrin systems, when with the help of manganese porphyrins, for example, or SOD mimic, hydrogen peroxide is accumulated in the system. Afterwards, the accumulated hydrogen peroxide is used in oxidation reactions of various substrates with iron porphyrin components of the catalyst. 

John Pedersen (Unizyme Laboratories) demonstrated how multienzyme systems can be used along with His affinity tags and subtractive IMAC to produce industrial enzymes in both packed or expanded bed adsorption systems. The efficiency of the approach was demonstrated with the produc-... 

---