**2. The central role of the pyruvate dehydrogenase (PDH) and alphaketoglutarate (KDGH) complexes in cell metabolism**

The evolution of the high-energy, oxygen-dependent metabolism of eukaryotes [4,5] has produced mitochondrial metabolic pathways whose control is centrally focused on a series of dehydrogenases. Two of these pivotal dehydrogenases, the pyruvate dehydrogenase (PDH) and alpha-ketoglutarate or 2-oxoglutarate (KDGH) complex will be our central focus here. These dehydrogenases control the entry of carbon into the TCA cycle from two major sources, carbohydrate and gluconeogenic amino acids (pyruvate; PDH) and glutamine (KGDH), the most abundant serum amino acid and a central carbon source for normal and pathological tissues (Figure 1). The TCA cycle, in turn, is almost exclusively responsible for the capture of reducing potential from nutrients for the purpose of driving the oxygendependent mitochondrial electron transport system for ATP production [6].

In the solid, three dimensional structure of animal bodies the availability of nutrients and molecular oxygen is locally contingent and dynamically variable. Thus, continuous real-time

© 2012 Bingham and Zachar, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

control of PDH and KGDH activities is essential to proper function. Moreover, in various pathological contexts these regulatory processes are substantially altered in ways that are essential to that pathology and this may reflect targets for therapeutic intervention (see sections 5 and 8 below for detailed references).

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 43

PDH decarboxylates pyruvate, releasing carbon dioxide, capturing reducing potential and transferring the two-carbon acyl unit (acetate) to Co-enzyme A (CoA) (Figure 2). Structurally homologous KGDH catalyzes the analogous reaction, decarboxylating alphaketoglutarate and generating succinyl-CoA. Each of these reactions is highly exergonic and also generates rapidly dispersed or consumed products (carbon dioxide and acyl-CoA). Thus, the initial decarboxylation reaction in each case is effectively irreversible, reflecting a

The catalytic details of PDH and KGDH are as follows (Figures 2 and 3). Each complex contains enzymatic activities conventionally referred to as E1, E2, and E3. E1 catalyzes oxidative decarboxylation (of pyruvate or alpha-ketoglutarate, respectively) using a thiamine pyrophosphate co-enzyme. The activated acyl unit created by this process is transferred to sulfhydryl groups of a lipoic acid (lipoate) residue of an E2 subunit. E2 lipoate is in the form of lipoamide, in which the carboxylic acid moiety of lipoate is joined in amide linkage to epsilon amino group of an E2 active site lysine. The two sulfurs on the lipoate moiety (Figures 3) exist in a disulfide configuration which is reduced and acylated (thioesters) to produce activated acetate or succinate residues. The E2 subunit further catalyzes the transfer of the lipoate-activated acyl residue to CoA leaving dihydrolipoamide. Finally, the E3 subunit catalyzes the transfer of the reducing potential in the dihydro-lipoamide residues of E2 to NAD+ (through enzyme disulfide to FAD to NAD steps) [7,8] to generate NADH for transfer of the reducing potential to the electron transport

Both PDH and KGDH are very large complexes containing multiple copies of each of these three key enzymatic activities (Figure 3). For example, mammalian PDH consists of 60 copies of the E2 subunit (48 of E2 itself and 12 of the E2 homolog called the E3 binding

This organization allows efficient channeling of the products of the intermediate reactions [10]. Flux through these complexes is subject to extensive regulation as expected from their central role in governing carbon flow in the face of variable supply and demand. The end products of PDH and KGDH activity, NADH and acyl-CoA, inhibit complex activity directly, apparently by binding to the E2 and E3 active sites, respectively [11,12]. However, PDH is much more extensively regulated through its associated kinases and

The four PDKs that regulate the mammalian PDH complex are a novel class of kinases apparently unrelated to the large families of serine-threonine and tyrosine kinases so prominent in other mammalian regulatory processes as reviewed in [7,8]. These kinases are named on the basis of their order of discovery, PDKs1-4. Though the PDKs represent a related family of kinases, the sequence divergence between the four different isoforms (61- 69%) is consistent with their significantly divergent regulatory behavior [14,15]. Moreover, PDK isoform function is apparently ancient and essential as the corresponding isoforms in

rodents and humans are at least 94% conserved as reviewed in [15].

forward commitment step.

protein, E3BP; Figure 3) [9].

phosphatases [13].

complex and regenerating oxidized lipoate (Figure 2).

**Figure 1.** The pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes govern the entry of carbohydrate- and glutamine-derived carbon, respectively, into the TCA cycle. Glucose and glutamine are two major carbon sources feeding many mammalian cells, including tumor cells. These molecules are metabolized to support ATP generation, through cytosolic glycolysis and oxygen-dependent mitochondrial metabolism in which most reducing potential is derived from the TCA cycle. As well, both carbon and reducing potential from glucose and glutamine are delivered to anabolic, biosynthetic functions. Among these are provision of citrate and NADPH for lipid biosynthesis as illustrated and delivery of carbon skeletons from glycolysis and the TCA cycle for amino acid biosynthesis (not illustrated). The entry of glucose derived carbon (either from dietary glucose or from amino acids used for gluconeogenesis) is controlled by the PDH complex and the entry of glutamine derived carbon is controlled by the KGCH complex. Figure redrawn from several sources, including [3].

PDH decarboxylates pyruvate, releasing carbon dioxide, capturing reducing potential and transferring the two-carbon acyl unit (acetate) to Co-enzyme A (CoA) (Figure 2). Structurally homologous KGDH catalyzes the analogous reaction, decarboxylating alphaketoglutarate and generating succinyl-CoA. Each of these reactions is highly exergonic and also generates rapidly dispersed or consumed products (carbon dioxide and acyl-CoA). Thus, the initial decarboxylation reaction in each case is effectively irreversible, reflecting a forward commitment step.

42 Dehydrogenases

including [3].

sections 5 and 8 below for detailed references).

control of PDH and KGDH activities is essential to proper function. Moreover, in various pathological contexts these regulatory processes are substantially altered in ways that are essential to that pathology and this may reflect targets for therapeutic intervention (see

**Figure 1.** The pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes govern the entry of carbohydrate- and glutamine-derived carbon, respectively, into the TCA cycle. Glucose and glutamine are two major carbon sources feeding many mammalian cells, including tumor cells. These molecules are metabolized to support ATP generation, through cytosolic glycolysis and oxygen-dependent mitochondrial metabolism in which most reducing potential is derived from the TCA cycle. As well, both carbon and reducing potential from glucose and glutamine are delivered to anabolic, biosynthetic functions. Among these are provision of citrate and NADPH for lipid

biosynthesis as illustrated and delivery of carbon skeletons from glycolysis and the TCA cycle for amino acid biosynthesis (not illustrated). The entry of glucose derived carbon (either from dietary glucose or from amino acids used for gluconeogenesis) is controlled by the PDH complex and the entry of glutamine derived carbon is controlled by the KGCH complex. Figure redrawn from several sources,

The catalytic details of PDH and KGDH are as follows (Figures 2 and 3). Each complex contains enzymatic activities conventionally referred to as E1, E2, and E3. E1 catalyzes oxidative decarboxylation (of pyruvate or alpha-ketoglutarate, respectively) using a thiamine pyrophosphate co-enzyme. The activated acyl unit created by this process is transferred to sulfhydryl groups of a lipoic acid (lipoate) residue of an E2 subunit. E2 lipoate is in the form of lipoamide, in which the carboxylic acid moiety of lipoate is joined in amide linkage to epsilon amino group of an E2 active site lysine. The two sulfurs on the lipoate moiety (Figures 3) exist in a disulfide configuration which is reduced and acylated (thioesters) to produce activated acetate or succinate residues. The E2 subunit further catalyzes the transfer of the lipoate-activated acyl residue to CoA leaving dihydrolipoamide. Finally, the E3 subunit catalyzes the transfer of the reducing potential in the dihydro-lipoamide residues of E2 to NAD+ (through enzyme disulfide to FAD to NAD steps) [7,8] to generate NADH for transfer of the reducing potential to the electron transport complex and regenerating oxidized lipoate (Figure 2).

Both PDH and KGDH are very large complexes containing multiple copies of each of these three key enzymatic activities (Figure 3). For example, mammalian PDH consists of 60 copies of the E2 subunit (48 of E2 itself and 12 of the E2 homolog called the E3 binding protein, E3BP; Figure 3) [9].

This organization allows efficient channeling of the products of the intermediate reactions [10]. Flux through these complexes is subject to extensive regulation as expected from their central role in governing carbon flow in the face of variable supply and demand. The end products of PDH and KGDH activity, NADH and acyl-CoA, inhibit complex activity directly, apparently by binding to the E2 and E3 active sites, respectively [11,12]. However, PDH is much more extensively regulated through its associated kinases and phosphatases [13].

The four PDKs that regulate the mammalian PDH complex are a novel class of kinases apparently unrelated to the large families of serine-threonine and tyrosine kinases so prominent in other mammalian regulatory processes as reviewed in [7,8]. These kinases are named on the basis of their order of discovery, PDKs1-4. Though the PDKs represent a related family of kinases, the sequence divergence between the four different isoforms (61- 69%) is consistent with their significantly divergent regulatory behavior [14,15]. Moreover, PDK isoform function is apparently ancient and essential as the corresponding isoforms in rodents and humans are at least 94% conserved as reviewed in [15].

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 45

**Figure 3.** Functional organization of the pyruvate dehydrogenase (PDH) complex in mammals. This complex catalyzes the oxidative decarboxylation of pyruvate to yield acetyl-CoA to feed carbohydrate carbon into the TCA cycle and to capture reducing potential in NADH for transfer to the electron transport complex (ETC) to support ATP synthesis during step down transfer to molecular oxygen (Figure 2). The mammalian PDH complex is built around a core consisting of the inner domains of ca. 48 copies of the catalytically active E2 protein and 12 copies of the catalytically inactive E2 homolog, E3BP, The E1 subunit binds to E2 and the E3 subunit binds to E3BP. Both E2 and E3BP contain lipoate residues joined in amide linkage to the epsilon amino groups of specific lysine residues in the lipoyl domains (L1-L3). The lipoamide residues on the two lipoyl domains of E2 and the single domain of E3BP are acylated, reduced and re-oxidized during the PDH catalytic cycle (Figure 2). The zig-zag lines indicate highly flexible proline-rich domains allowing the E2 subunits high mobility throughout the complex and, perhaps, beyond. The regulatory kinases (PDKs) control PDH activity by phosphorylating (inactivating) the E1 subunit of the complex. Their activity is antagonized by two phosphatases (PDPs). The status of E2 lipoate residues strongly modulates the activity of the PDKs with acylation/reduction stimulating PDK activity. Figure redrawn from several sources, including [7].The PDKs normally function as dimers [see, for example 16,17]. Of particular potential importance is the observation that at least two of the PDK isoforms (PDK1 & 2) readily form heterodimers [18]. This raises the possibility that there might be as many as 10 different PDK isoforms (four homodimers and six heterodimers). In view of the substantial changes in PDK levels associated with malignancy (below), this potential

The PDKs regulate PDH complex activity by responding to diverse allosteric modulators. High ratios of acetyl-CoA to CoASH and NADH to NAD+ represent signals of saturation of mitochondrial demand. Elevations of these ratios are strong allosteric activators of PDKs,

complexity may be very important and is worthy of more investigation.

**Figure 2.** The catalytic cycle of the pyruvate dehydrogenase (PDH) complex. This cycle begins with the oxidative decarboxylation of pyruvate catalyzed by the E1 subunit, generating carbon dioxide and an activated two carbon unit (hydroethyl thiamine pyrophosphate, HE-TPP) as illustrated at the 10 o'clock position. E1 then catalyzes the transfer of this two carbon unit to one of the lipoate residues of the E2 subunits, creating acyl-lipoamide as illustrated at the 12 o'clock position. The E2 catalytic activity transfers this two carbon unit to CoA, creating acetyl-CoA and dihydro-lipoamide as illustrated at the 4 o'clock position. Finally, E3 catalyzes the transfer of the dihydro-lipoamide to NADH (through protein sulfhydryl and FAD intermediates) to recreate the oxidize form, lipoamide, as illustrated at the 8 o'clock position. Both the E2 catalyzed transfer of the lipoamide acyl group to CoA and the E3 catalyzed transfer of dihydro-lipoamide reducing potential to NADH are highly reversible reactions. Figure redrawn from several sources, including [8].

**Figure 2.** The catalytic cycle of the pyruvate dehydrogenase (PDH) complex. This cycle begins with the oxidative decarboxylation of pyruvate catalyzed by the E1 subunit, generating carbon dioxide and an activated two carbon unit (hydroethyl thiamine pyrophosphate, HE-TPP) as illustrated at the 10 o'clock position. E1 then catalyzes the transfer of this two carbon unit to one of the lipoate residues of the E2 subunits, creating acyl-lipoamide as illustrated at the 12 o'clock position. The E2 catalytic activity transfers this two carbon unit to CoA, creating acetyl-CoA and dihydro-lipoamide as illustrated at the 4 o'clock position. Finally, E3 catalyzes the transfer of the dihydro-lipoamide to NADH (through protein sulfhydryl and FAD intermediates) to recreate the oxidize form, lipoamide, as illustrated at the 8 o'clock position. Both the E2 catalyzed transfer of the lipoamide acyl group to CoA and the E3 catalyzed transfer of dihydro-lipoamide reducing potential to NADH are highly reversible reactions. Figure

redrawn from several sources, including [8].

**Figure 3.** Functional organization of the pyruvate dehydrogenase (PDH) complex in mammals. This complex catalyzes the oxidative decarboxylation of pyruvate to yield acetyl-CoA to feed carbohydrate carbon into the TCA cycle and to capture reducing potential in NADH for transfer to the electron transport complex (ETC) to support ATP synthesis during step down transfer to molecular oxygen (Figure 2). The mammalian PDH complex is built around a core consisting of the inner domains of ca. 48 copies of the catalytically active E2 protein and 12 copies of the catalytically inactive E2 homolog, E3BP, The E1 subunit binds to E2 and the E3 subunit binds to E3BP. Both E2 and E3BP contain lipoate residues joined in amide linkage to the epsilon amino groups of specific lysine residues in the lipoyl domains (L1-L3). The lipoamide residues on the two lipoyl domains of E2 and the single domain of E3BP are acylated, reduced and re-oxidized during the PDH catalytic cycle (Figure 2). The zig-zag lines indicate highly flexible proline-rich domains allowing the E2 subunits high mobility throughout the complex and, perhaps, beyond. The regulatory kinases (PDKs) control PDH activity by phosphorylating (inactivating) the E1 subunit of the complex. Their activity is antagonized by two phosphatases (PDPs). The status of E2 lipoate residues strongly modulates the activity of the PDKs with acylation/reduction stimulating PDK activity. Figure redrawn from several sources, including [7].The PDKs normally function as dimers [see, for example 16,17]. Of particular potential importance is the observation that at least two of the PDK isoforms (PDK1 & 2) readily form heterodimers [18]. This raises the possibility that there might be as many as 10 different PDK isoforms (four homodimers and six heterodimers). In view of the substantial changes in PDK levels associated with malignancy (below), this potential complexity may be very important and is worthy of more investigation.

The PDKs regulate PDH complex activity by responding to diverse allosteric modulators. High ratios of acetyl-CoA to CoASH and NADH to NAD+ represent signals of saturation of mitochondrial demand. Elevations of these ratios are strong allosteric activators of PDKs, shutting down PDH activity in response to demand saturation [19, 20, 21]. Though saturation of mitochondrial demand is most often produced in response to high fat or carbohydrate intake in healthy animals, as reviewed in [8], the altered metabolism of tumor cells also creates a very new metabolic environment wherein these regulatory processes may be entrained for different purposes (below).

The Pyruvate Dehydrogenase Complex in Cancer:

Implications for the Transformed State and Cancer Chemotherapy 47

binding domains, but also each of these domains is separated by highly flexible

These details will be important to us below in considering the possible mechanisms of action to pursue in developing new drugs based on lipoate structure. Specifically, each PDK can potentially interact simultaneously with two lipoate residues, as well as rapidly migrate between residues. This creates an environment in which PDKs may effectively sample the lipoate cloud, regulating their activity in response to what is effectively a measurement of the aggregate cloud status, especially the acylation/redox status of lipoate as determined by both forward flux through the complex and by backward charging of lipoate from NADH

Such a sampling procedure would yield especially robust assessment of enzyme saturation and, thus, allow PDK control of PDH to be more reliably connected to the global mitochondrial energy status. *In vitro* studies demonstrate that free lipoate can interact with the PDH (and KGDH) complexes [29]. Thus, we anticipate that exogenous lipoate analogs might be effective in modulating the activity of PDK bound to the PDH complex (below).

**3. Clinical implications of the reconfiguration of metabolism in cancer** 

The hypothesis that metabolism may be altered in important ways in cancer is longstanding [30]. However, our understanding of cancer metabolism and its relationship to malignancy and clinical outcome has only recently begun to receive extensive attention. Though our knowledge about cancer metabolism remains quite incomplete, some important global

First, fluxes of both glucose and glutamine, two major carbon sources, are substantially upregulated in many tumors as reviewed in [31-36]. More specifically, mutational changes altering signaling through the Akt pathway enhance glucose uptake and glycolytic metabolism [37]. Likewise, alterations of Myc expression substantially up-regulate

Second, evidence from multiple perspectives supports the view that one of the major functions of these tumor-specific changes is the redirection of metabolism toward provision of increased levels of anabolic substrates. For example, large fractions of glucose carbon are diverted into the pentose phosphate pathway in support of nucleotide synthesis and into amino acid biosynthesis. Likewise, a large portion of glutamine carbon is siphoned off of the TCA cycle as citrate to support lipid biosynthesis (including fatty acids and sterols) (Figure

The therapeutic implications of the alterations in cancer metabolism depend heavily on how unique these changes are to tumor cells. There is good evidence that some elements of the anabolic shift are not unique to tumors cells, but may also be shared with rapidly dividing normal cells. For example, activated lymphocytes show some of these same metabolic

(alanine/proline-rich) linker domain.

and acetyl-CoA products (above).

**cells** 

insights have emerged.

glutamine utilization [3, 38, 39].

1). We can call this the *anabolic shift* in tumor cells.

PDKs are also subject to allosteric regulation by pyruvate [22] . Elevated pyruvate levels interact synergistically with ADP to inhibit PDK activity (activating PDH). This PDK inhibition apparently results from the binding of pyruvate to PDK-ADP inhibiting exchange for ATP and, thus, phosphorylation. Dichloroacetate (DCA) is a pyruvate analog and acts very similarly to pyruvate in this allosteric modulation of PDK activity [22] DCA was subsequently investigated as an anticancer drug (below) on the basis of this observation.

The final relevant feature of PDK regulation is the response of these regulatory kinases to the lipoyl domains of the E2 subunits of PDH [7, 8, 23]. PDK1-3 bind strongly to the L2 lipoate domain of E2 and more weakly to the E2 L1 lipoyl domain (Figure 3). PDK4 binds to the L1 E2 domain and to the sole lipoyl domain of E3BP (Figure 3). This lipoyl domain binding requires the lipoamide component of the domain. Moreover, the well characterized binding of PDK3 to the E2 L2 domain defines the binding site in detail including the amino acids interacting with the lipoamide residue [16]. These residues are highly conserved in PDK structure [17] indicating that this binding mode is likely universal to the PDKs. This binding of PDKs to E2 lipoyl domains juxtaposes them to their E1 target (associated with the E2 subunit binding domain; Figure 3), producing a large increase in the kinase reaction rate. PDK dimers apparently interact simultaneously with two lipoyl domains, producing very high binding affinity to the PDH complex and supporting hand-over-hand migration of the PDKs through the complex [24].

Most importantly for our purposes here, the reduction and acylation state of the E2 lipoates strongly modulates associated PDK activity [25, 26]. Specifically, acylation and reduction of lipoate enhances PDK2 activity [7,8, 27]. Moreover, acetyl-CoA and NADH can be used as substrates to run elements of the PDH reaction (above) in reverse, thereby creating reduced and/or acylated lipoate residues in response to elevation in the levels of acetyl-CoA and NADH in the mitochondrial matrix [25,26]. In other words, the lipoate residues of PDH represent a real-time census of these crucial features of the energy status of the mitochondrial matrix.

Several additional details of lipoate regulation of PDH and KGDH are also relevant for this discussion. The lipoates in PDH and KGDH comprise a dense cloud of highly mobile residues [28], potentially interacting with crucial regulatory functions. This condition results from several features of the structure of these complexes. PDH illustrates the crucial issues. Each of the ca. 48 E2 subunits contains 2 lipoate (lipoamide) residues and each of the 12 E3BP proteins contains 1 such residue, for a total of 108 lipoate residues in each complex. The regulatory kinases (PDKs-1-4) interact with these lipoate residues embedded in the lipoyl domains (Figure 3). Moreover, these lipoyl domains not only have the boom-like structure resulting from their connection to lysine epsilon amino groups in the E3 lipoyl binding domains, but also each of these domains is separated by highly flexible (alanine/proline-rich) linker domain.

46 Dehydrogenases

be entrained for different purposes (below).

PDKs through the complex [24].

mitochondrial matrix.

shutting down PDH activity in response to demand saturation [19, 20, 21]. Though saturation of mitochondrial demand is most often produced in response to high fat or carbohydrate intake in healthy animals, as reviewed in [8], the altered metabolism of tumor cells also creates a very new metabolic environment wherein these regulatory processes may

PDKs are also subject to allosteric regulation by pyruvate [22] . Elevated pyruvate levels interact synergistically with ADP to inhibit PDK activity (activating PDH). This PDK inhibition apparently results from the binding of pyruvate to PDK-ADP inhibiting exchange for ATP and, thus, phosphorylation. Dichloroacetate (DCA) is a pyruvate analog and acts very similarly to pyruvate in this allosteric modulation of PDK activity [22] DCA was subsequently investigated as an anticancer drug (below) on the basis of this observation.

The final relevant feature of PDK regulation is the response of these regulatory kinases to the lipoyl domains of the E2 subunits of PDH [7, 8, 23]. PDK1-3 bind strongly to the L2 lipoate domain of E2 and more weakly to the E2 L1 lipoyl domain (Figure 3). PDK4 binds to the L1 E2 domain and to the sole lipoyl domain of E3BP (Figure 3). This lipoyl domain binding requires the lipoamide component of the domain. Moreover, the well characterized binding of PDK3 to the E2 L2 domain defines the binding site in detail including the amino acids interacting with the lipoamide residue [16]. These residues are highly conserved in PDK structure [17] indicating that this binding mode is likely universal to the PDKs. This binding of PDKs to E2 lipoyl domains juxtaposes them to their E1 target (associated with the E2 subunit binding domain; Figure 3), producing a large increase in the kinase reaction rate. PDK dimers apparently interact simultaneously with two lipoyl domains, producing very high binding affinity to the PDH complex and supporting hand-over-hand migration of the

Most importantly for our purposes here, the reduction and acylation state of the E2 lipoates strongly modulates associated PDK activity [25, 26]. Specifically, acylation and reduction of lipoate enhances PDK2 activity [7,8, 27]. Moreover, acetyl-CoA and NADH can be used as substrates to run elements of the PDH reaction (above) in reverse, thereby creating reduced and/or acylated lipoate residues in response to elevation in the levels of acetyl-CoA and NADH in the mitochondrial matrix [25,26]. In other words, the lipoate residues of PDH represent a real-time census of these crucial features of the energy status of the

Several additional details of lipoate regulation of PDH and KGDH are also relevant for this discussion. The lipoates in PDH and KGDH comprise a dense cloud of highly mobile residues [28], potentially interacting with crucial regulatory functions. This condition results from several features of the structure of these complexes. PDH illustrates the crucial issues. Each of the ca. 48 E2 subunits contains 2 lipoate (lipoamide) residues and each of the 12 E3BP proteins contains 1 such residue, for a total of 108 lipoate residues in each complex. The regulatory kinases (PDKs-1-4) interact with these lipoate residues embedded in the lipoyl domains (Figure 3). Moreover, these lipoyl domains not only have the boom-like structure resulting from their connection to lysine epsilon amino groups in the E3 lipoyl These details will be important to us below in considering the possible mechanisms of action to pursue in developing new drugs based on lipoate structure. Specifically, each PDK can potentially interact simultaneously with two lipoate residues, as well as rapidly migrate between residues. This creates an environment in which PDKs may effectively sample the lipoate cloud, regulating their activity in response to what is effectively a measurement of the aggregate cloud status, especially the acylation/redox status of lipoate as determined by both forward flux through the complex and by backward charging of lipoate from NADH and acetyl-CoA products (above).

Such a sampling procedure would yield especially robust assessment of enzyme saturation and, thus, allow PDK control of PDH to be more reliably connected to the global mitochondrial energy status. *In vitro* studies demonstrate that free lipoate can interact with the PDH (and KGDH) complexes [29]. Thus, we anticipate that exogenous lipoate analogs might be effective in modulating the activity of PDK bound to the PDH complex (below).
