Enzymes multienzyme complexes

Enzymes Various separate enzymes Multienzyme complex... 

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. 

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. 

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. 

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. 

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.

Nature, however, has performed more than simple stepwise transformations using a combination of enzymes in so-called multienzyme complexes, it performs multistep synthetic processes. A well-known example in this context is the biosynthesis of fatty acids. Thus, Nature can be quoted as the inventor of domino reactions. Usually, as has been described earlier in this book, domino processes are initiated by the application of an organic or inorganic reagent, or by thermal or photochemical treatment. The use of enzymes in a flask for initiating a domino reaction is a rather new development. One of the first examples for this type of reaction dates back to 1981 [3], although it should be noted that in 1976 a bio-triggered domino reaction was observed as an undesired side reaction by serendipity [4]. 

Each reaction of p oxidation is catalyzed by a different enzyme. Chemically, they re pretty much the same as the reverse of the individual reaction of fatty acid synthesis, with two exceptions (1) p oxidation uses FAD for the formation of the double bond at the C-2 position, and (2) the reactions occur with the fatty acid attached to CoA rather than to the pantetheine of a multienzyme complex. 

While PAL itself is recovered from the soluble fractions of cellular lysates via ultracentrifugation purifications, strong experimental evidence suggests it exists as part of a multienzyme complex associated with the endoplasmic reticulum (ER), forming a metabolic channel tunneling substrates directly from one enzymatic reactive site to another (Winkel 2004). The enzyme physically anchored to the ER is cinnamate... 

The PDHC catalyzes the irreversible conversion of pyruvate to acetyl-CoA (Fig. 42-3) and is dependent on thiamine and lipoic acid as cofactors (see Ch. 35). The complex has five enzymes three subserving a catalytic function and two subserving a regulatory role. The catalytic components include PDH, El dihydrolipoyl trans-acetylase, E2 and dihydrolipoyl dehydrogenase, E3. The two regulatory enzymes include PDH-specific kinase and phospho-PDH-specific phosphatase. The multienzyme complex contains nine protein subunits, including... 

Each turn of the P-oxidation spiral splits off a molecule of acetyl-CoA. The process involves four enzymes catalysing, in turn, an oxidation (to form a double bond), a hydration, another oxidation (forming a ketone from a secondary alcohol) and the transfer of an acetyl group to coenzyme A (Figure 7.12). The process of P-oxidation operates as a multienzyme complex in which the intermediates are passed from one enzyme to the next, i.e. there are no free intermediates. The number of molecules of ATP generated from the oxidation of one molecule of the long-chain fatty acid pal-mitate (C18) is given in Table 7.4. Unsaturated fatty acids are also oxidised by the P-oxidation process but require modification before they enter the process (Appendix 7.3). 

More recently, Kutney and co-workers (220) have investigated whether the same dihydropyridinium intermediate 109 is involved in the enzymatic conversion of catharanthine (4) and vindoline (3) to anhydrovinblastine (8) as is involved in the chemical conversion. Use of a cell-free preparation from a 5-day culture of the AC3 cell line gave 18% of the bisindole alkaloids leurosine (11), Catharine (10), vinamidine (25), and hydroxy-vinamidine (110), with 10 predominating. When the incubations were carried out for only 5-10 min, the dihydropyridinium intermediate was detected followed by conversion to the other bisindole alkaloids, with FAD and MnClj required as cofactors. Clearly a multienzyme complex is present in the supernatant, but further purification led to substantial loss of enzymatic activity. The chemical formation of anhydrovinblastine (3) is carried out with catharanthine A-oxide (107), but when this compound was used in the enzyme preparation described, no condensation with vindoline (3) occurred to give bisindole alkaloids. This has led Kutney and co-workers to suggest (220) that the A-oxide 108 is not an intermediate in the biosynthetic pathway, but rather that a 7-hydroperoxyindolenine... 

The pyruvate dehydrogenase reaction takes place in the mitochondrial matrix (see p. 210). Three different enzymes [E1-E3] form the PDH multienzyme complex (see B). 

T0293 Enzyme Technologies, Inc., Dissolved Oxygen In Sim Treatment (DO IT) T0294 Enzyme Technologies, Inc., Multienzyme Complex (MZC)... 

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