Protein multienzyme complexes

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. 

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. 

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. 

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. 

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.]... 

What structural features of biotin and lipoic acid allow these cofactors to be covalently bound to a specific protein in a multienzyme complex yet participate in reactions at active sites on other enzymes of the complex ... 

Acetyl-CoA carboxylase of E. coli is a multienzyme complex that consists of three protein components that can be isolated individually Biotin carboxyl carrier protein (BCCP), biotin carboxylase, and carboxyltransferase (fig. 

After malonyl-CoA synthesis, the remaining steps in fatty acid synthesis occur on fatty acid synthase, which exists as a multienzyme complex. In the initial reactions acetyl-CoA and malonyl-CoA are transferred onto the protein complex by acetyl-CoA transacylase and malonyl-CoA transacylase (step 1 and step 2 in fig. 18.12a). The acceptor for the acetyl and malonyl groups is acyl carrier protein (ACP). ACP also carries all of the intermediates during fatty acid biosynthesis. The prosthetic group that binds these intermediates is... 

Outline of the reactions for fatty acid biosynthesis. Fatty acids grow in steps of two-carbon units and take place on a multienzyme complex, (a) The initial reactions of fatty acid biosynthesis are shown. In the first reaction, acetyl-CoA reacts with ACP (acyl carrier protein) to form acetyl-ACP (step 1). ACP is shown with its SH group emphasized (see fig. 18.13) to remind readers that the acyl derivatives are linked to ACP via a thioester bond. Malonyl-CoA, derived from the carboxylation of acetyl-CoA (see fig. 18.9), reacts... 

Fatty acid synthesis takes place in eight steps. All except the first step take place on a multienzyme complex. The intermediates on this complex are carried by attachment of the acid group in thioester linkage to phosphopantetheine of the acyl carrier protein (ACP). The multienzyme complex greatly increases the efficiency of fatty acid synthesis, because for each step in the pathway the next enzyme is always near at hand, and the dilution of intermediates is minimized. 

In many cases the amino acid pathway branches so that two or more amino acids are formed. Aspartate is the precursor of four other amino acids found in proteins Isoleucine, threonine, methionine, and lysine (see fig. 21.2). The first step in this overall pathway entails the conversion of aspartate to /3-aspartyl-phosphate by aspartokinase. One might imagine that all four of the amino acid end products of this pathway would act together to inhibit this enzyme. However, in E. coli a different solution has been found. In this bacterium there are three aspartokinases which appear to be parts of different multienzyme complexes leading to threonine and leucine for aspartokinase I, methionine for aspartokinase II and lysine for aspartokinase III. As might be expected threonine and isoleucine inhibit aspartokinase I,... 

A detailed study of amino acid sequences and mechanistic similarities in various polyketide synthase (PKS) enzymes has led to two main types being distinguished. Type I enzymes consist of one or more large multifunctional proteins that possess a distinct active site for every enzyme-catalysed step. On the other hand, Type II enzymes are multienzyme complexes that carry out a single set of repeating activities. Like fatty acid synthases, PKSs catalyse the condensation of coenzyme A esters of simple carboxylic acids. However, the variability at each step in... 

RN Perham. Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes a paradigm in the design of a functional protein. Biochemistry 30 8501-8512, 1991. 

Fatty acid synthase (FAS) carries out the chain elongation steps of fatty acid biosynthesis. FAS is a large multienzyme complex. In mammals, FAS contains two subunits, each containing multiple enzyme activities. In bacteria and plants, individual proteins, which associate into a large complex, catalyze the individual steps of the synthesis scheme. 

---