Introduction

The bulk of ATP used by many cells to maintain homeostasis is produced by the oxidation of Pyr in TCAC. During this oxidation process, NADH and FADH2 are generated that are used to drive the processes of OPP, which are responsible for converting the reducing potential of NADH and FADH2 to the high energy Pi in ATP.

The fate of Pyr depends on the cell energy charge. In cells or tissues with a high energy charge Pyr is directed toward gluconeogenesis, but when the energy charge is low Pyr is preferentially oxidized to CO2 and H2 in TCAC, with generation of 15 Eqs ATP per Pyr. The enzymatic activities of TCAC (and of OPP) are located in the mitochondrion. When transported into the mitochondrion, Pyr encounters two principal metabolizing enzymes: PC and PDH, the first enzyme of PDHC. With a high cell-energy charge CoA is highly acylated, principally as Ac-CoA, and able allosterically to activate PC, directing Pyr toward gluconeogenesis. When the energy charge is low CoA is not acylated, PC is inactive, and Pyr is preferentially metabolized via PDHC and the enzymes of TCAC to CO2 and H2. Reduced NADH and FADH2 generated during the oxidative reactions can then be used to drive ATP synthesis via OPP.

 PDHC

The reactions of PDHC serves to interconnect the metabolic pathways of glycolysis, gluconeogenesis and fatty acid synthesis to TCAC. The importance of PDHC to the maintenance of homeostasis is evident from the fact that although diseases associated with deficiencies of PDHC have been observed, affected individuals often do not survive to maturity. Since the energy metabolism of highly aerobic tissues such as the brain is dependent on normal conversion of Pyr to Ac-CoA, aerobic tissues are most sensitive to deficiencies in components of PDHC. Most genetic diseases associated with PDHC deficiency are due to mutations in PDH. The main pathologic result of such mutations is moderate to severe cerebral lactic acidosis and encephalopathies.

PDHC catalyzes 5 sequential reactions (Fig. 1) that lead to the oxidative decarboxylation of Pyr with formation of CO2, Ac-CoA, and NADH. The overall stoichometry of the whole process is:

Pyr + CoA + NAD+ ------> CO2 + Ac-CoA + NADH + H+

PDHC is comprised of (Fig. 5):

The E1 and E3 dimers surround the core of PDHC, which is made by all 60 E2 subunits that are arranged to form dodecahedron. PDHC also requires 5 different coenzymes: CoA, NAD+, FAD, lipoic acid and TPP. 3 of the coenzymes are tightly bound to enzymes of PDHC (TPP, Lipoic acid - which is joined in amide linkage to the e-amino group of a Lys residue and is altogether called lipoyllysyl arm, and FAD) and 2 are employed as carriers of the products of PDHC activity (CoA and NAD+). The lipoyllysyl arm serves as a long tether which channels the reaction intermediates between the different subunits of PDHC.

 Reactions

Reaction 1

E1 is a TPP-requiring α2β2 heterotetramer with two active sites that interact with each other during catalysis. Pyr is decarboxylated by E1, with the intermediate formation of hydroxyethyl-TPP (steps 1 and 2 in Fig. 1).

There are 3 specific Ser in the α subunit of E1 that are subject to ATP-dependent PP-tion and inactivation by PDK. PDP dephosphorylates these 3 Ser and reactivates PDC.

Reaction 2

The hydroxyethyl group is transferred to the next enzyme, E2. The reaction occurs by attack of the hydroxyethyl group carbanion on the lipoamide disulfide, followed by the elimination of TPP from the intermediate adduct to form acetyl-dihydrolipoamide and regenerate active E1. The hydroxyethyl carbanion is thereby oxidized to an acetyl group by the concomitant reduction of the lipoamide disulfide bond (steps 3 and 4 in Fig. 1).

Reaction 3

E2 catalyzes the transfer of the acetyl group to CoA, yielding Ac-CoA and dihydrolipoamide-E2. This is a transesterification in which the sulfhydryl group of CoA attacks the acetyl group of acetyl-dihydrolipoamide-E2 to form a tetrahedral intermediate, which decomposes to Ac-CoA and dihydrolipoamide-E2 (step 5 in Fig. 1).

Reaction 4

E3 reoxidizes dihydrolipoamide, thereby completing the catalytic cycle of E2. Oxidized E3 contains a reactive disulfide group and a tightly bound FAD. The oxidation of dihydrolipoamide is a disulfide interchange reaction. The lipoamide disulfide bond forms with concomitant reduction of E3ís reactive disulfide to 2 sulfhydryl groups.

Reaction 5

Reduced E3 is reoxidized by NAD+. The E3ís active sulfhydryl groups are reoxidized by the enzyme-bound FAD, which is thereby reduced to FADH2. FADH2 is then reoxidized to FAD by NAD+, producing NADH.

During the course of the reaction the acetyl group derived from decarboxylation of Pyr is bound to TPP . The next reaction of the complex is the transfer of the 2--carbon acetyl group from acetyl-TPP to lipoic acid, the covalently bound coenzyme of DLAT. The transfer of the acetyl group from acyl-lipoamide to CoA results in the formation of 2 sulfhydryl (SH) groups in lipoate requiring reoxidation to the disulfide (S-S) form to regenerate lipoate as a competent acyl acceptor. DLD with FAD as a cofactor, catalyzes that oxidation reaction. The final activity of PDHC is the transfer of reducing equivalents from the FADH2 of dihydrolipoyl dehydrogenase to NAD+. The fate of the NADH is oxidation via mitochondrial electron transport, to produce 3 Eqs ATP.

 Regulation

The decarboxylation of Pyr by E1 is irreversible and since there are no other pathways in mammals for the synthesis of Ac-CoA from Pyr it is crucial that the reaction is carefully controlled. 2 regulatory systems are employed:

Product  inhibition

They compete with NAD+ and CoA for the binding site on the respective enzymes. They also drive the reversible transacetylase (E2) and dihydrolipoyl dehydrogenase (E3) reactions backwards. High ratios of [NADH]/[NAD+] and [Ac-CoA]/[CoA] therefore maintain E2 in the acetylated form, incapable of accepting the hydroxyethyl group from TPP on E1. This, in turn, ties up TPP on E1 in its hydroxyethyl form, decreasing the rate of Pyr-decarboxylation.

Phosphorylation / Dephosphorylation

PDK inactivates E1 by catalyzing the PP-tion of a specific PDH-Ser. Hydrolysis of this phospho-Ser by PDP reactivates the complex. The products of the E1-reaction, NADH and Ac-CoA, in addition to their direct effect on PDHC, activate PDK. Since NADH and Ac-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDH kinase activity, reinforcing down-regulation of PDH activity in energy-rich cells.The resultant PP-tion inactivates the complex just as the products themselves inhibit it. INS is involved in the control of this system through its indirect activation of PDH-phosphatase. In cardiac muscle PDH activity is increased by catecholamines.

PDK functions as either homodimer or heterodimer (build from PDK1 and PDK2 subunits 1). Although PDKs phosphorylate specific serines of the E1α subunit, they show very little amino acid sequence similarity with eukaryotic Ser/Thr protein kinases. PDKs belong to the ATPase/kinase superfamily based on similarity of their catalytic domains. PDKs are bound to the PDHC core through the lipoyl domains of E2, and this interaction is important for their efficient catalytic function. PDK activity is enhanced through changes in the status of the lipoyl domains from oxidized to reduced and acetylated forms, which in turn depend on the NADH/NAD+ and Ac-CoA/CoA ratios. PDK isoenzymes differ in their catalytic activity, responsiveness to the modulators such as NADH and Ac-CoA, and tissue-specific expression. PDK1 is present mostly in heart, whereas PDK2 is found in most tissues. PDK3 is predominantly expressed in testis, whereas heart and skeletal muscle have the highest amount of PDK4 (Fig. 2).

PDK1 is one of the 4 mitochondrial enzymes (PDK1, PDK2, PDK3, and PDK4) responsible for regulation of the PDHC and, consequently, aerobic oxidation of carbohydrate fuels in general. PDK1 is present mostly in heart, whereas PDK2 is found in most tissues. PDK3 is predominantly expressed in testis, whereas heart and skeletal muscle have the highest amount of PDK4. Although PDKs phosphorylate specific serines (site 1, Ser264; site 2, Ser271; site 3, Ser203) of the E1α subunit, they show very little amino acid sequence similarity with eukaryotic Ser/Thr protein kinases. PDKs were suggested to belong to the ATPase/kinase superfamily based on similarity of their catalytic domains. PDKs are bound to the PDHC core through the lipoyl domains of E2, and this interaction is important for their efficient catalytic function. PDK activity is enhanced through changes in the status of the lipoyl domains from oxidized to reduced and acetylated forms, which in turn depend on the NADH/NAD+ and Ac-CoA/CoA ratios.

PDK isoenzymes differ in their catalytic activity, responsiveness to the modulators such as NADH and acetyl-CoA, and tissue-specific expression. PDKs also differ in their activity toward the PP-tion sites of the E1α subunit which is as follows - for site 1, PDK2 > PDK4=PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1; site 3 is phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase is demonstrated by PDK3. The activity of the four PDKs is stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, doesn't affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Thus site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of PDHC in normal and pathophysiological states. 4

Pyr, ADP, NAD+, CoA, Ca2+ (high Mg2+) and K+ inhibit PDH-kinase while Ca2+ and Mg2+ activate PDH-phosphatase.

2 products of PDHC, NADH and Ac-CoA, are negative allosteric effectors on PDH-a, the non-phosphorylated, active form of PDH. These effectors reduce the affinity of the enzyme for Pyr, thus limiting the flow of carbon through PDHC. However, Pyr is a potent negative effector on PDKs, with the result that when Pyr levels rise, PDH-a will be favored even with high levels of NADH and Ac-CoA.

[Pyr] which maintain PDH in the active form (PDH-a) are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting Pyr to Ac-CoA. With large amounts of Pyr in cells having high energy charge and high NADH, Pyr carbon will be directed to the 2 main storage forms of carbon -glycogen via gluconeogenesis and fat production via fatty acid synthesis - where Ac-CoA is the principal carbon donor.

Although the regulation of PDH-b phosphatase is not well understood, it is quite likely regulated to maximize Pyr oxidation under energy-poor conditions and to minimize PDH activity under energy-rich conditions (Fig. 3).

Boxes

DLD is a subunit of 3 different members of the a-keto-acid dehydrogenase family of multienzyme complexes , namely - PDHC , OGDC and BCKDC