A -Oxoacid dehydrogenases

Figure 15-15 Sequence of reactions catalyzed by a-oxoacid dehydrogenases. The substrate and product are shown in boxes, and the path of the oxidized oxoacid is traced by the heavy arrows. The lipoic acid "head" is shown rotating about the point of attachment to a core subunit. However, a whole flexible domain of the core is also thought to move.

The a-oxoacid dehydrogenases yield CoA derivatives which may enter biosynthetic reactions. Alternatively, the acyl-CoA compounds may be cleaved with generation of ATP. The pyruvate formate-lyase system also operates as part of an ATP-generating system for anaerobic organisms, for example, in the "mixed acid fermentation" of enterobacteria such as E. coli (Chapter 17). These two reactions, which are compared in Fig. 15-16, constitute an important pair of processes both of which accomplish substrate-level phosphorylation. They should be compared with the previously considered examples of substrate level phosphorylation depicted in Eq. 14-23 and Fig. 15-16. 

The intermediary metabolism has multienzyme complexes which, in a complex reaction, catalyze the oxidative decarboxylation of 2-oxoacids and the transfer to coenzyme A of the acyl residue produced. NAD" acts as the electron acceptor. In addition, thiamine diphosphate, lipoamide, and FAD are also involved in the reaction. The oxoacid dehydrogenases include a) the pyruvate dehydrogenase complex (PDH, pyruvate acetyl CoA), b) the 2-oxoglutarate dehydrogenase complex of the tricarboxylic acid cycle (ODH, 2-oxoglutarate succinyl CoA), and c) the branched chain dehydrogenase complex, which is involved in the catabolism of valine, leucine, and isoleucine (see p. 414). 

The PDH complex of the bacterium Escherichia coli has been particularly well studied. It has a molecular mass of 5.3 10 , and with a diameter of more than 30 nm it is larger than a ribosome. The complex consists of a total of 60 polypeptides (1, 2) 24 molecules of E2 (eight trimers) form the almost cube-shaped core of the complex. Each of the six surfaces of the cube is occupied by a dimer of E3 components, while each of the twelve edges of the cube is occupied by dimers of El molecules. Animal oxoacid dehydrogenases have similar structures, but differ in the numbers of subunits and their molecular masses. 

Many other oligomeric enzymes and other complex assemblies of more than one kind of protein subunit are known. For example, the 2-oxoacid dehydrogenases are huge 2000- to 4000-kDa complexes containing three different proteins with different enzymatic activities in a cubic array (Fig. 15-14). The filaments of striated muscle (Chapter 19), antibodies and complement of blood (Chapter 31), and the tailed bacteriophages (Box 7-C ) all have complex molecular architectures. 

NAD+ serves as the oxidant. The reaction is catalyzed by a complex of enzymes whose molecular mass varies from 4 to 10 x 106, depending on the species and exact substrate.297 Separate oxoacid dehydrogenase systems are known for pyruvate,298-300 2-oxoglut-arate,301 and the 2-oxoacids with branched side chains derived metabolically from leucine, isoleucine, and... 

Within many tissues the enzymatic activities of the pyruvate and branched chain oxoacid dehydrogenases complexes are controlled in part by a phosphorylation -dephosphorylation mechanism (see Eq. 17-9). Phosphorylation of the decarboxylase subunit by an ATP-dependent kinase produces an inactive phosphoenzyme. A phosphatase reactivates the dehydrogenase to complete the regulatory cycle (see Eq. 17-9 and associated discussion). The regulation is apparently accomplished, in part, by controlling the affinity of the protein for... 

In a rare autosomal recessive condition (discovered in 1954) the urine and perspiration has a maple syrup odor/ High concentrations of the branched-chain 2-oxoacids formed by transamination of valine, leucine, and isoleucine are present, and the odor arises from decomposition products of these acids. The branched-chain amino acids as well as the related alcohols also accumulate in the blood and are found in the urine. The biochemical defect lies in the enzyme catalyzing oxidative decarboxylation of the oxoacids, as is indicated in Fig. 24-18. Insertions, deletions, and substitutions may be present in any of the subunits (Figs. 15-14,15-15). The disease which may affect one person in 200,000, is usually fatal in early childhood if untreated. Children suffer seizures, mental retardation, and coma. They may survive on a low-protein (gelatin) diet supplemented with essential amino acids, but treatment is difficult and a sudden relapse is apt to prove fatal. Some patients respond to administration of thiamin at 20 times the normal daily requirement. The branched-chain oxoacid dehydrogenase from some of these children shows a reduced affinity for the essential coenzyme thiamin diphosphate.d... 

There are two 2-oxoacid dehydrogenase multienzyme complexes in E. coli. One is specific for pyruvate, the other for 2-oxoglutarate. Each complex is about the size of a ribosome, about 300 A across. The pyruvate dehydrogenase is composed of three types of polypeptide chains El, the pyruvate decarboxylase (an a2 dimer of Mr — 2 X 100 000) E2, lipoate acetyltransferase (Mr = 80 000) and E3, lipoamide dehydrogenase (an a2 dimer of Mr = 2 X 56 000). These catalyze the oxidative decarboxylation of pyruvate via reactions 1.6, 1.7, and 1.8. (The relevant chemistry of the reactions of thiamine pyrophosphate [TPP], hydroxyethylthiamine pyrophosphate [HETPPJ, and lipoic acid [lip-S2] is discussed in detail in Chapter 2, section C3.)... 

The absence in halobacteria of the oxoacid dehydrogenase complexes creates another puzzle. In most known systems, the role of the enzyme lipoamide dehydrogenase is to reoxidize the lipoic acid that is involved in the oxidation of the oxoacids in the oxoacid dehydrogenase complexes. This enzyme was nonetheless found in H. halobium and purified to homogeneity by Danson et al. (1986). What, then, is its function It is likely that lipoamide dehydrogenase assumes a different role in halobacteria. Another reducing system unique to... 

As reviewed previously [1], there are distinct mechanistic similarities between the archaebacterial oxidoreductases and the 2-oxoacid dehydrogenase complexes, despite their obvious structural differences. Thus, both systems form acyl-CoA via a TPP-... 

This compound is the product of oxidation of the enamine by any one of the following oxidizing agents on enzymes most commonly by the dithiolane ring of lipoic acid covalently amidated to a lysine side chain in the 2-oxoacid dehydrogenase multienzyme complexes less frequently by FAD in the pyruvate oxidases — these come in two flavors, forming acetate in Escherichia coli and forming acetylphosphate in L. plantarum, finally by NAD. ... 

Scheme 6 (a)Domain structure of the E2 component of the 2-oxoacid dehydrogenase complexes, (b) Schematic layout of domains in the 1-lipolyl E2ec. The lipoyl domain (LD) with the appended lipoamide visits the active sites of all three components of PDHc-ec. 

Amino acid catabohsm is particularly important dining starvation. Because of the mass of muscle, amino acid catabohsm is particularly important in this tissue which, in starvation, supplies the liver with most of its gluconeogenic precursors (see also Fig. 13-11). Amino acids resulting from proteolysis during starvation are interconverted in the muscle so that 60% of the amino acid mass that leaves the muscle is either glutamine or alanine. The branched-chain amino acids valine, leucine, and isoleucine, which are aU essential amino acids, are deaminated in muscle by a specific aminotransferase, and the corresponding 2-oxoacids are transported to the liver for further metabohsm via branched-chain 2-oxoacid dehydrogenase (BCOADH). The aminotransferase is inactive in the hver, and this ensures that the peripheral tissues are supphed with valine, leucine, and isoleucine. 

Figure 11.2 Reaction sequences catalyzed by 2-oxoacid dehydrogenase complex Pyruvate dehydrogenase complex (PDC) and a-ketoglutarate dehydrogenase complex (aKGDC) catalyze the oxidative decarboxylation of pyruvate (R = CH3) and a-ketoglutarate (R = CH2CH2COOH) to Acetyl-CoA and succinyl CoA respectively. Three component enzymes 2-oxoacid (pyruvate/a-ketoglutarate) decarboxylase, lipoate acetyltransferase/succinyltransferase, dihydrolipoate dehydrogenase as well as five cofactors, namely (1) thiamine pyrophosphate (TPP) and its acylated form, (2) lipoamide (LipS2), reduced form and acylated form, (3) flavin adenine dinucleotide (FAD) and its reduced form, (4) nicotinamide adenine dinucleotide (NAD ) and its reduced form, and (5) coenzyme A (CoASH) and its acylated product are involved.

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