Multienzyme

One of the most fascinating recent developments in biology has been the discovery of numerous highly complex biopolymer assemblies (see also section C2.14.2.3) such as the ribosome or the bacterial flagellum [93, 94 and 95], the envy of nanoteclmologists seeking to miniaturize man-made mechanical devices (note that the word machinery is also sometimes used to refer to multienzyme complexes such as the proteasome [96]), and an entire... 

In biological systems molecular assemblies connected by non-covalent interactions are as common as biopolymers. Examples arc protein and DNA helices, enzyme-substrate and multienzyme complexes, bilayer lipid membranes (BLMs), and aggregates of biopolymers forming various aqueous gels, e.g, the eye lens. About 50% of the organic substances in humans are accounted for by the membrane structures of cells, which constitute the medium for the vast majority of biochemical reactions. Evidently organic synthesis should also develop tools to mimic the Structure and propertiesof biopolymer, biomembrane, and gel structures in aqueous media. 

Multienzyme Electrodes. Coupling the reactions of two or more immobilized enzymes increases the number of analytes that can be measured. An electro-inactive component can be converted by an enzyme to a substrate that is subsequentiy converted by a second enzyme to form a detectable end product (57). For example, a maltose [69-79-4] sensor uses the enzymes glucoamylase and glucose oxidase, which convert... 

Multienzyme electrodes can increase sensitivity from micromolar to nanomolar detection levels (53,57). In this case the substrate is converted to a detectable product by one enzyme, then that product is recycled into the initial substrate by another enzyme resulting in an amplification of the response signal. For example, using lactate oxidase and lactate dehydrogenase immobilized in poly(vinyl chloride), an amplification of 250 was obtained for the detection oflactate (61). 

Hyde, C.C., et al. Three-dimensional structure of the tryptophan synthase az pz multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263 17857-17871, 1988. 

As research reveals the ultrastructural organization of the cell in ever greater detail, more and more of the so-called soluble enzyme systems are found to be physically united into functional complexes. Thus, in many (perhaps all) metabolic pathways, the consecutively acting enzymes are associated into stable multienzyme complexes that are sometimes referred to as metabolons, a word meaning units of metabolism. ... 

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. 

Lipoic acid is an acyl group carrier. It is found in pyruvate dehydrogenase zard a-ketoglutarate dehydrogenase, two multienzyme complexes involved in carbohydrate metabolism (Figure 18.34). Lipoie acid functions to couple acyl-group transfer and electron transfer during oxidation and decarboxylation of a-keto adds. 

Rittenberg and Bloch showed in the late 1940s that acetate units are the building blocks of fatty acids. Their work, together with the discovery by Salih Wakil that bicarbonate is required for fatty acid biosynthesis, eventually made clear that this pathway involves synthesis of malonyl-CoA. The carboxylation of acetyl-CoA to form malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids (Figure 25.2). The reaction is catalyzed by acetyl-CoA carboxylase, which contains a biotin prosthetic group. This carboxylase is the only enzyme of fatty acid synthesis in animals that is not part of the multienzyme complex called fatty acid synthase. 

The enzymes that catalyze formation of acetyl-ACP and malonyl-ACP and the subsequent reactions of fatty acid synthesis are organized quite differently in different organisms. We first discuss fatty acid biosynthesis in bacteria and plants, where the various reactions are catalyzed by separate, independent proteins. Then we discuss the animal version of fatty acid biosynthesis, which involves a single multienzyme complex called fatty acid synthase. 

Fatty Acid Synthesis in Eukaryotes Occurs on a Multienzyme Complex... 

In bacteria, each step in fatty-acid sjmthesis is catalyzed by separate enzymes. In vertebrates, however, fatty-acid synthesis is catalyzed by a large, multienzyme complex called a synthase that contains two identical subunits of 2505 amino acids each and catalyzes all steps in the pathway. An overview of fatty-acid biosynthesis is shown in Figure 29.5. 

In bacteria, ACP is a small protein of 77 residues that transports an acyl group from enzyme to enzyme. In vertebrates, however, ACP appears to be a long arm on a multienzyme synthase complex, whose apparent function is to shepherd an acyl group from site to site within the complex. As in acetyl CoA, the acyl group in acetyl ACP is linked by a thioester bond to the sulfur atom of phosphopantetheine. The phosphopantetheine is in turn linked to ACP through the side-chain -OH group of a serine residue in the enzyme. 

Following the formation of malonyl CoA, another nucleophilic acyl substitution reaction occurs in step 4 to form the more reactive malonyl ACP, thereby binding the malonyl group to an ACP arm of the multienzyme synthase. At this point, both acetyl and malonyl groups are bound to the enzyme, and the stage is set for their condensation. 

Further chain elongation of palmitic acid occurs by reactions similar to those just described, but CoA rather than ACP is the carrier group, ancl separate enzymes are needed for each step rather than a multienzyme synthase complex. 

Die synthetische Fahigkeit ganzer Enzym-Systeme oder von Multienzym-Komplexen kann geniitzt werden. 

This thermodynamic driving force is particularly useful tvith multienzyme equilibrium systems such as that used in the gram-scale synthesis of tv ro equivalents ofo-xylulose 5-phosphate (104) from (26) (Figure 10.38) [171,172]. Similarly, the corresponding 1-deoxy-D-xylulose 5-phosphate tvas efficiently produced from pyruvate and (34) by the catalytic action of the thiamine diphosphate-dependent 1-deoxy-D-xylulose 5-phosphate synthase (DXS) (EC 2.2.1.7) from E. coli [173]. 

The nanostructured molecular arrangements from DNA developed by Seeman may find applications as biological encapsulation and drug-delivery systems, as artificial multienzymes, or as scaffolds for the self-assembling nanoscale fabrication of technical elements. Moreover, DNA-protein conjugates may be anticipated as versatile building blocks in the fabrication of multifunctional supramolecular devices and also as highly functional-... 

Pyruvate is oxidized to acetyl-GoA by a multienzyme complex, pyruvate dehydrogenase, that is dependent on the vitamin cofactor thiamin diphosphate. 

Behai RH et al Regulation of the pyruvate dehydrogenase multienzyme complex. Annu Rev Nutr 1993 13 497. 

Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of acetyl-CoA to mal-onyl-CoA in the presence of ATP and acetyl-CoA carboxylase. Acetyl-CoA carboxylase has a requirement for the vitamin biotin (Figure 21-1). The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and transcarboxylase, as well as a regulatory allosteric site. The reaction takes place in two steps (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl to acetyl-CoA to form malonyl-CoA. 

In bacteria and plants, the individual enzymes of the fatty acid synthase system are separate, and the acyl radicals are found in combination with a protein called the acyl carrier protein (ACP). However, in yeast, mammals, and birds, the synthase system is a multienzyme polypeptide complex that incorporates ACP, which takes over the role of CoA. It contains the vitamin pantothenic acid in the form of 4 -phosphopan-tetheine (Figure 45-18). The use of one multienzyme functional unit has the advantages of achieving the effect of compartmentalization of the process within the cell without the erection of permeability barriers, and synthesis of all enzymes in the complex is coordinated since it is encoded by a single gene. 

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys— SH, cysteine thiol.) The— SH of the 4 -phosphopantetheine of one monomer is in close proximity to the— SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a "head-to-tail" arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

Acetyl-CoA carboxylase is required to convert acetyl-CoA to malonyl-CoA. In turn, fatty acid synthase, a multienzyme complex of one polypeptide chain with seven separate enzymatic activities, catalyzes the assembly of palmitate from one acetyl-CoA and seven malonyl-CoA molecules. 

Gencic S, DA Grahame (2003) Nickel in subunit P of the acetyl-CoA decarboxylase/synthase multienzyme complex in methanogens. J Biol Chem 278 6101-6110. 

Allen JR, SA Ensign (1997) Characterization of three protein components required for functional reconstitution of the epoxide carboxylase multienzyme complex from Xanthobacter strain Py2. J Bacterial 179 3110-3115. 

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. 

Guatelli, J. C et al. (1990). Isothermal in vitro amplification of nucleic acids by multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87,1874-1878. 

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