Proteins multienzyme

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. 

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, 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. 

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-... 

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.

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. 

Delcroix, M., Sajid, M., Caffrey, C.R., Lim, K.-C., Dvorak, J., Hsieh, I., Bahgat, M., Dissous, C., and McKerrow, J.H. (2006) A multienzyme network functions in intestinal protein digestion by a platy-helminth parasite./. Biol. Chem. 281, 39316-39329. 

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... 

Fatty add synthase is a large multienzyme complex in the cytoplasm that is rapidly induced in the liver after a meal by high carbohydrate and the concomitant rise in insulin levels. It contains an acyl carrier protein (AGP) that requires the vitamin pantothenic add. Althoi malonyl CoA is the substrate used by fetty acid synthase, only the carbons from the acetyl CoA portion are actually incorporated into the fatty acid produced. Therefore, the fetty add is derived entirely from acetyl CoA. 

Increasing evidence indicates that accumulation of aberrant or misfolded proteins, protofibril formation, ubiquitin-proteasome system dysfunction, and the direct or indirect consequences of abnormal protein aggregation and accumulation represent deleterious events linked to neurodegeneration (255,256). Ubiquitination is an essential cellular process affected by a multienzyme cascade involving Els (ubiquitin-activating enzymes), E2s (ubiquitin-conjugation enzymes or UBCs), and E3s (ubiquitin-protein Ugases) (12,257) (see Fig. 10.4). 

Specialized Purification Procedures Purification of membrane proteins, 182, 499 purification of integral membrane proteins, 104, 329 reconstitution of membrane proteins, 104, 340 purification of DNA-binding proteins by site-specific DNA affinity chromatography, 182, 521 purification of glycoproteins, 182, 529 purification of multienzyme complexes, 182, 539. 

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... 

Detailed mechanisms of the Ub/proteasome-dependent multienzyme machines that target a protein substrate and then processively destroy it remain to be understood. The findings described above (1) identified a class of interactions between the targeting and proteolytic branches of the Ub system that does not involve a multi-Ub chain. 

The former is a protein of 14.7 kDa involved in the multienzyme nucleotide excision repair (NER) pathway with a determined NMR solution structure . In this protein, the Zn + possesses rather a structural than a catalytic role. Zn NMR spectra were acquired using a rather sophisticated probe (for details, see Reference 87) and operating at temperatures 5-250 K. Data acquisition was performed with the application of spin-echo methods for enhanced sensitivity . Specifically, experiments were carried out at 25 K using a combination of CP (cross-polarization) and spikelet echo pulse sequences which provide a considerable increase in signal-to-noise ratio (of the order of 30) relative to a classical quadrupole echo pulse sequence. The proton field strength applied to the above measurements was 60 kHz with a matching field of 20 kHz for zinc and a contact time... 

The control of enzyme activity by the environment of a polyatomic framework is a vast topic, which I shall not attempt to cover fully in this report. Instead I will concentrate on some selected interactions between and within polypeptide chains that influence enzymatic activity. First, elementary steps involved in ligand-protein, intraprotein, and interprotein interactions are considered. Then enzymes consisting of a single polypeptide chain are discussed, followed by enzymes consisting of multiple polypeptide chains. The concluding sections are concerned with multienzyme complexes and enzymes associated with membranes. 

The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain. For fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associated with the inner mitochondrial membrane, the trifunctional protein (TFP). TFP is a heterooctamer of 4/34 subunits. Each a subunit contains two activities, the enoyl-CoA hydratase and the /3-hydroxyacyl-CoA dehydrogenase the /3 subunits contain the thiolase activity. This tight association of three enzymes may allow efficient substrate channeling from one active site to the... 

Bocanegra, J. H. A.,Scrutton,N. S. Perham, R. N. (1993). Creation of an NADP-dependent pyruvate dehydrogenase multienzyme complex by protein engineering. Biochemistry, 32, 2737-40. 

The remaining series of reactions of fatty acid synthesis in eukary-l otes is catalyzed by the multifunctional, dimeric enzyme, fatty acid synthase. Each fatty acid synthase monomer is a multicatalytic polypeptide with seven different enzymic activities plus a domain that covalently binds a molecule of 4 -phosphopantetheine. [Note 4-Phosphopantetheine, a derivative of the vitamin pantothenic add (see p. 379), carries acetyl and acyl units on its terminal thiol (-SH)j group during fatty acid synthesis. It also is a component of 00-enzyme A.] In prokaryotes, fatty acid synthase is a multienzyme complex, and the 4 -phosphopantetheine domain is a separate protein, referred to as the acyl carrier protein (ACP). ACP is used below to refer to the phosphopantetheine-binding domain of the eukaryotic fatty acid synthase molecule. The reaction numbers in1 brackets below refer to Figure 16.9. [Note The enzyme activities listed are actually separate catalytic domains present in each mulf-1 catalytic fatty acid synthase monomer.]... 

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