Multi-enzyme complexes

Thiamin has a central role in energy-yielding metabo-hsm, and especially the metabohsm of carbohydrate (Figure 45-9). Thiamin diphosphate is the coenzyme for three multi-enzyme complexes that catalyze oxidative decarboxylation reactions pymvate dehydrogenase in carbohydrate metabolism a-ketoglutarate dehydro-... 

For a soluble enzyme that is not part of a multi-enzyme complex, the fastest rate of enzyme-inhibitor association is determined by the rate of molecular collisions between the two binding partners (i.e., the enzyme and the inhibitor) in solution. The rate of molecular collisions is in turn controlled by the rate of diffusion. The diffusion-limited rate of molecular collisions is dependent on the radii of the two binding molecules and the solution temperature and viscosity (Fersht, 1999) ... 

Nature gives us some illustrative examples of iterative methodologies in its biochemical mechanisms. The fatty acid-polyketide biosynthesis is one of them. The assembly of acyl units by sequential Claisen-type condensations to form a polyketide or fatty acid takes place at a multi-enzyme complex, at which the initial molecule is lengthened by one C2-unit per pass of a reaction cycle (Fig. 2). 

Domino reactions are not a new invention - indeed, Nature has been using this approach for billions of years However, in almost of Nature s processes different enzymes are used to catalyze the different steps, one of the most prominent examples being the synthesis of fatty acids using a multi-enzyme complex starting from acetic acid derivatives. 

The biosynthesis of polyketides (including chain initiation, elongation, and termination processes) is catalyzed by large multi-enzyme complexes called polyketide synthases (PKSs). The polyketides are synthesized from starter units such as acetyl-CoA, propionyl-CoA, and other acyl-CoA units. Extender units such as malonyl-CoA and methylmalonyl-CoA are repetitively added via a decarboxylative process to a growing carbon chain. Ultimately, the polyketide chain is released from the PKS by cleavage of the thioester, usually accompanied by chain cyclization [49]. 

STAFFORD, H.A., Possible multi-enzyme complexes regulating the formation of Cg-C3 phenolic compounds and lignins in higher plants, Rec. Adv. Phytochem., 1974, 8, 53-79. 

This is not the only example of Nature inventing the assembly line a long time before Henry Ford—both pyru-vate dehydrogenase and a-ketoglutarate dehydrogenase, mentioned earlier in the chapter, are also multi-enzyme complexes. 

Once an enzyme-catalysed reaction has occurred the product is released and its engagement with the next enzyme in the sequence is a somewhat random event. Only rarely is the product from one reaction passed directly onto the next enzyme in the sequence. In such cases, enzymes which catalyse consecutive reactions, are physically associated or aggregated with each other to form what is called a multi enzyme complex (MEC). An example of this arrangement is evident in the biosynthesis of saturated fatty acids (described in Section 6.30). Another example of an organized arrangement is one in which the individual enzyme proteins are bound to membrane, as for example with the ATP-generating mitochondrial electron transfer chain (ETC) mechanism. Intermediate substrates (or electrons in the case of the ETC) are passed directly from one immobilized protein to the next in sequence. 

PDH is a multi-enzyme complex consisting of three separate enzyme units pyruvate decarboxylase, transacetylase and dihydrolipoyl dehydrogenase. Serine residues within the decarboxylase subunit are the target for a kinase which causes inhibition of the PDH the inhibition can be rescued by a phosphatase. The PDH kinase (PDH-K) is itself activated, and the phosphatase reciprocally inhibited, by NADH and acetyl-CoA. Figure 3.12(a and b) show the role and control of PDH. 

Fatty acid and triglyceride (triacylglycerol) synthesis acetyl-CoA carboxylase and fatty acid synthase multi-enzyme complex... 

The fatty acid synthesis pathway can be seen to occur in two parts. An initial priming stage in which acetyl-CoA is converted to malonyl-CoA by a carboxylation reaction (Figure 6.9) is followed by a series of reactions which occur on a multi-enzyme complex (MEC), which achieves chain elongation forming C16 palmitoyl-CoA. The whole process occurs in the cytosol. 

The FAS multi-enzyme complex synthesizes saturated C16 fatty acids, but cells and tissues need unsaturated and longer chain fatty acids. The palmitoyl-CoA can be modified by either chain elongation and/or oxidation in order to produce different fatty acid molecules. Both elongation and desaturation occur within the smooth endoplasmic reticulum (SER, microsomal fraction) of the cell. 

Winkel BSJ (2009) Metabohte channehng and multi-enzyme complexes. In Osbourn AE, Lanzotti V (eds) Plant-derived natural products synthesis, function, and appUcation. Springer, New York... 

Flavanone 3 -hydroxylase (F3 H ECl.14.13.21 CYP75B) activity was initially identified in microsomal preparations of golden weed (Haplopappus gracilis) [110]. E3 H from irradiated parsley cell cultures was later biochemically analyzed and characterized as a cytochrome P450 having an absolute requirement for NADPH and molecular oxygen as cofactors [111]. The enzyme has been shown to have activity with flavanones, flavones, dihydroflavonols, and flavonols, but does not appear to have activity with anthocyanidins [111]. The first cDNA clone for E3 H was isolated from Petunia [112]. It has been suggested that E3 H may serve as an anchor for the proposed flavonoid multi-enzyme complex on the cytosolic surface of the endoplasmic reticulum [44]. 

A Fatty acids are constmcted by stepwise addition of two-carbon units by a large multi-enzyme complex located in the cytoplasm of all cells. 

C. Fatty acid synthase is a large multi-enzyme complex that catalyzes the addition of two-carbon units in a seven-step cycle (Figure 8-2). 

It is impossible to describe and explain enzyme kinetics unless is explained by an entire book therefore, this chapter describes only briefly some aspects. It is strongly recommended to read once more a textbook on enzymology and enzyme kinetics. Especially the reaction kinetics of enzyme oligomeres, multi-enzyme complexes, and phenomena of cooperation are too complex to explain in just a few pages. 

RNA polymerase II represents a multi-enzyme complex of at least 12 proteins, but whose exact composition is difficult to determine. Hiis is due to the instabUity of the holo-complex, which makes the pmification and characterization of the enzyme difficult. Furthermore, it is likely that multiple forms of RNA polymerase II exist, each of slightly different composition and performing different functions. 

Escherichia coli (strain B [28] overproducing strain JM83(pKT8P3) [31] infected and uninfected [32] infected by bacteriophage T4, host-coded enzyme from infected E. coli is part of bacteriophage T4 dNTP-synthesiz-ing multi-enzyme complex [25]) [25, 28, 31, 32]... 

PN Lowe, JA Hodgson, RN Perham. Dual role of single multi enzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis. Biochem J 215 133-140, 1983. 

CF Hawkins, A Borges, RN Perham. Cloning and sequence analysis of the genes encoding the a and (3 subunits of the El component of the pyruvate dehydrogenase multi enzyme complex of Bacillus stearothermophilus. Eur JBiochem 191 337-346, 1990. 

The biosynthesis of polyketides is analogous to the formation of long-chain fatty acids catalyzed by the enzyme fatty acid synthase (FAS). These FASs are multi-enzyme complexes that contain numerous enzyme activities. The complexes condense coenzyme A (CoA) thioesters (usually acetyl, propionyl, or malonyl) followed by a ketoreduction, dehydration, and enoylreduction of the [3-keto moiety of the elongated carbon chain to form specific fatty acid products. These subsequent enzyme activities may or may not be present in the biosynthesis of polyketides. 

Very long chain fatty acids are initially oxidized in the peroxisome where the initial oxidation step is catalyzed by acyl-CoA oxidase and the subsequent steps in fS-oxidation are catalyzed by a multi-enzyme complex with hydratase, dehydo-genase, and thiolase activities. Unsaturated fatty acids require additional enzymatic activities, including enoyl-CoA isomerase and dienoyl-CoA reductase. Readers are directed to Vance and Vance (2) for additional details regarding fi-oxidation, including the details of the metabolic reactions. 

The second metabolic pathway which we have chosen to describe is the tricarboxylic acid cycle, often referred to as the Krebs cycle. This represents the biochemical hub of intermediary metabolism, not only in the oxidative catabolism of carbohydrates, lipids, and amino acids in aerobic eukaryotes and prokaryotes, but also as a source of numerous biosynthetic precursors. Pyruvate, formed in the cytosol by glycolysis, is transported into the matrix of the mitochondria where it is converted to acetyl CoA by the multi-enzyme complex, pyruvate dehydrogenase. Acetyl CoA is also produced by the mitochondrial S-oxidation of fatty acids and by the oxidative metabolism of a number of amino acids. The first reaction of the cycle (Figure 5.12) involves the condensation of acetyl Co and oxaloacetate to form citrate (1), a Claisen ester condensation. Citrate is then converted to the more easily oxidised secondary alcohol, isocitrate (2), by the iron-sulfur centre of the enzyme aconitase (described in Chapter 13). This reaction involves successive dehydration of citrate, producing enzyme-bound cis-aconitate, followed by rehydration, to give isocitrate. In this reaction, the enzyme distinguishes between the two external carboxyl groups... 

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