Dihydrolipoyl transacetylase

Diamine oxidase (pig kidney, human pregnancy plasma, pea seedlings) 261 Dihydrolipoyl transacetylase (Escherichia coli)[27] Dipeptidyl carboxypeptidase[281 DNA polymerase-a (human)1291... 

Pyruvate dehydrogenase is a MEC consisting of three separate catalytic proteins (i) a component with combined pyruvate decarboxylase/dehydrogenase activity (ii) a dihydrolipoyl transacetylase (also called acetyl transferase) unit and (iii) linked dihy-drolipoyl dehydrogenase. This is clearly a very big protein the pyruvate decarboxylase/... 

Dihydrolipoyl transacetylase transfers the acetyl CoA to its lipoic acid... 

Figure 7-1. Conversion of pyruvate to acetyl CoA by the pyruvate dehydrogenase complex. The three enzymes, pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase, exist in a complex associated with the mitochondrial matrix. Each enzyme requires at least one coenzyme that participates in the reaction. TPP, thiamine pyrophosphate Lip, lipoic acid CoA, coenzyme A.

The pyruvate dehydrogenase complex from Escherichia coli is considerably more complex than tryptophan synthetase. It has a molecular weight of approximately 4.6 millon and contains three enzymes pyruvate dehydrogenase (Et), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3).82 The overall reaction catalyzed by the complex is... 

FIGURE 16-4 Lipoic acid (lipoate) in amide linkage with a Lys residue. The lipoyllysyl moiety is the prosthetic group of dihydrolipoyl transacetylase (E2 of the PDH complex). The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group. 

The PDH complex contains three enzymes—pyruvate dehydrogenase (EJ, dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3)—each present in multiple copies. The number of copies of each enzyme and therefore the size of the complex varies among species. The PDH complex isolated from mammals is about 50 nm in diameter—more than five times the size of an entire ribosome and big enough to be visualized with the electron microscope (Fig. 16-5a). In the bovine enzyme, 60 identical copies of E2 form a pentagonal dodecahedron (the core) with a diameter of about 25 nm (Fig. 16-5b). (The core of the Escherichia coli enzyme contains 24 copies of E2.) E2 is the point of... 

The PDH complex is composed of multiple copies of three enzymes pyruvate dehydrogenase, Ei (with its bound cofactor TPP) dihydrolipoyl transacetylase, E2 (with its covalently bound lipoyl group) and dihydrolipoyl dehydrogenase, E3 (with its cofactors FAD and NAD). 

The hydroxyethyl intermediate is oxidized by transfer to the disulfide form of lipoic acid covalently bound to dihydrolipoyl transacetylase. —... 

The conversion of pyruvate to acetyl-CoA. The reactions are catalyzed by the enzymes of the pyruvate dehydrogenase complex. This complex has three enzymes pyruvate decarboxylase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. In addition, five coenzymes are required thiamine pyrophosphate, lipoic acid, CoASH, FAD, and NAD+. Lipoic acid is covalently attached to... 

Dihydrolipoyl transacetylase core (each spherical enzyme unit contains 3 identical subunits)... 

Dihydrolipoyl transacetylase (Ef) catalyzes this reaction. The energy-rich thioester bond is preserved as the acetyl group is transferred to CoA. Recall that CoA serves as a carrier of many activated acyl groups, of which acetyl is the simplest (Section 14.3.1). Acetyl CoA, the fuel for the citric acid cycle, has now been generated from pyruvate. 

Dihydrolipoyl transacetylase E2 24 Lipoamide Transfer of the acetyl group to CoA... 

The lipoamide of dihydrolipoyl transacetylase constitutes a long arm which may now move the acetyl group from the active site of pyruvate DH to its own active site where the lipoamide is exchanged for Coenzyme A-SH. (On the mammalian enzyme the 60 subunits of the transacetylase seem to form a pool of lipoyl groups among which the acetyl groups are freely exchanged). 

Note that in the reactions of dihydrolipoyl transacetylase the lipoamide has been reduced from a disulphide to two sulphhydryl groups. In order to continue operation lipoamide must be reoxidized and that is accomplished by the final enzyme of the complex, dihydrolipoyl dehydrogenase. The reactions catalyzed by this enzyme are complex, but the net result is the transfer of two electrons from the lipoamide to NAD+ to give NADH. 

Lipoamide Lipoic acid is linked through an amide bond to the side-chain NH2 group of a lysine residue in dihydrolipoyl transacetylase... 

A detailed mechanism of the first two reactions is shown in Figure 13-8. In the third step, catalyzed by dihydrolipoyl transacetylase, the acetyl group attached to E2 is transferred to CoASH, with formation of the dithiol form of the lipoyl group and acetyl-CoA. 

In the second step, also catalyzed by pyruvate dehydrogenase, the hydroxyethyl group is transferred to the oxidized form of the lipoyl-lysyl prosthetic group of dihydrolipoyl transacetylase (E2) ... 

Regulation of pyruvate dehydrogenase (PD) by inactivation and reactivation by a non-cAMP-dependent phosphorylation-dephosphorylation cycle. Although PD kinase phosphorylates three specific seryl residues in the a-subunit of PD, phosphorylation at any of these sites inactivates PD. The kinase and the phosphatase are under the influence of several regulators, and the dephospho-active PD is also regulated by end products. 0 = Activation 0 = inhibition E2 = dihydrolipoyl transacetylase E3 = dihydrolipoyl dehydrogenase. 

Despite the apparent simplicity of this highly exergonic reaction (AG° = -33.5 kJ/mol), its mechanism is one of the most complex known. The pyruvate dehydrogenase complex is a large multienzyme structure that contains three enzyme activities pyruvate dehydrogenase (Ej), also known as pyruvate decarboxylase, dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Each enzyme activity is present in multiple copies. Table 9.3 summarizes the number... 

Dihydrolipoyl transacetylase (E2) Catalyzes transfer of acetyl group to CoASH 24 (60) Lipoic acid, CoASH... 

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