The biochemistry of the pyruvate dehydrogenase complex*

In the Westernized world the daily dietary caloric requirements are roughly provided as follows: 40% from carbohydrates, 40% from fats, and 20% from proteins. In some populations in developing countries the daily caloric contribution from carbohydrate is even higher due to its readily available sources and relatively low cost. Glucose, the principal product of carbohydrate digestion, passes through a series of enzymatic steps first in the non-oxidative glycolytic pathway (from glucose to pyruvate) followed by efficient oxidative metabolism via the tricarboxylic acid cycle (from acetyl-CoA to CO2 and H2O) to harness a portion of its potential energy as ATP. Interestingly, these two major pathways are directly linked by the pyruvate dehydrogenase complex (PDC)11 localized in the mitochondrial matrix (Fig. 1). In the fed state acetyl-CoA generated from pyruvate (derived mostly from glucose and some dietary amino acids) is also utilized for the biosynthesis of lipids such as long chain fatty acids and cholesterol by lipogenic tissues (such as liver and adipose tissues and under special conditions in mammary glands during lactation and in the brain during the prenatal and early postnatal development). Additionally, amino acids from excess dietary protein are metabolized by several specialized reactions or pathways generating intermediates that have to be ultimately converted to pyruvate first and then to acetyl-CoA via PDC either for complete oxidation to CO2 and H2O or for lipogenesis. PDC is the only known reaction in most eukaryotes to generate acetyl-CoA (two-carbon compound) from pyruvate (three-carbon compound). Since this is a physiologically irreversible reaction and since there is no other known reaction or pathway to convert the two-carbon compound to pyruvate (or its equivalent) for the synthesis of glucose in animals, the flux through PDC is tightly regulated to meet the specific metabolic and energetic needs of different tissues during the fed and fasting (starvation) states. This is accomplished by covalent modification of the rate-limiting component of the complex involving sophisticated interplay among the components of the complex and allosteric modulations by acetyl-CoA and NADH, the products of the reaction (and also of fatty acid oxidation). It is evident from these simple considerations that PDC plays a key role as a gatekeeper of both caloric and glucose homeostasis in mammals. In this review, we will discuss recent developments concerning the structure-function relationship of this multienzyme complex from various organisms with emphasis on regulatory aspects of the mammalian complex. Detailed accounts of various aspects of this complex can be found in several excellent reviews [1–11]. PDC is present in most prokaryotic and eukaryotic organisms. It catalyzes several sequential reactions of oxidative decarboxylation of pyruvic acid by the action of its three catalytic components: (i) pyruvate dehydrogenase (E1) catalyzing the decarboxylation of pyruvate followed by reductive acetylation of lipoyl moieties covalently linked to the dihydrolipoamide acetyltransferase (E2), the second catalytic component of the complex; (ii) E2 catalyzing the formation of acetyl-CoA; and (iii) dihydrolipoamide dehydrogenase (E3) reoxidizing the reduced lipoyl moieties of E2 with the consequent reduction of NAD+ to NADH (Fig. 2) [12]. PDC is a multienzyme complex with a well organized structure that has two different morphologies based on symmetry of the central E2 core, i.e. octahedral for Gram-negative bacteria and icosahedral for eukaryotes (mammals, yeast, plants, and nematodes) and some Gram-positive bacteria. E2 consists of three well defined domains connected by flexible linkers: (i) inner domains interacting noncovalently with each other to form the core of the complex and each having the catalytic site; (ii) subunit-binding domain interacting with E1 and E3 (for PDC from bacteria, plants, and nematodes); and (iii) the lipoyl domain (containing up to three repeating units, one for yeast and Gram-positive bacteria, two for mammals, and three for Gram-negative bacteria; Fig. 3) providing coupling of the E1, E2, and E3 active sites by transferring an acetyl group and reducing equivalents. The core of Gram-negative bacterial PDC is composed of inner domains of 24 subunits of E2 and binds 24 dimers of E1(α2) and 12 dimers of E3(α2) (Table I). PDCs with icosahedral symmetry have 60-mer E2 cores that bind up to 30 tetramers of E1(α2β2) and 12 dimers of E3. Eukaryotic organisms have an additional component in PDC not present in bacterial complexes, i.e. E3-binding protein (BP) [6, 12]. BP has a domain structure similar to E2 structure except: (i) only one lipoyl domain is present; (ii) the subunit-binding domain binds E3 and not E1; and (iii) the inner domain does not perform a catalytic function (due to the absence of the critical histidine residue). The PDC core of eukaryotes is composed of the inner domains of E2 and BP [13]. Ascaris suum PDC has a BP without a lipoyl domain and only in the anaerobic form of this nematode [14]. Mammalian, plant, and nematode PDCs are regulated by reversible phosphorylation/dephosphorylation that requires additional regulatory enzymes, pyruvate dehydrogenase kinase (PDK), and phosphopyruvate dehydrogenase phosphatase (PDP) (Table I). The three-dimensional structures of several components of PDC were determined by x-ray crystallography: E1 from Escherichia coli [15] and human2 ; E1β subunit from Pyrobaculum aerophilum [6]; cubic core of E2 (consisting of the inner domains) from Azotobacter vinelandii, Bacillus stearothermophilus, and Enterococcus faecalis; subunit-binding domain of E2 from B. stearothermophilus; E2 lipoyl domains from B. stearothermophilus, E. coli, and A. vinelandii; and E3 from yeast, A. vinelandii, Pseudomonas putida, and Pseudomonas fluorescens [5, 6, 16]. So far it is not possible to solve the three-dimensional structure of the whole PDC because of its huge molecular mass of 8–10 million Da, hollow structure of its inner core with high solvent content, and flexibility of E2 domains. However, the structures of the E2 core and entire PDC from yeast and mammals were studied by cryoelectron microscopy (Fig. 4) [17, 18]. Both electron microscopy and crystal structures of the E2 core revealed the trimer building blocks in the cubic (8 trimers) or dodecahedral (20 trimers) structures. The trimers are connected by bridges and form a structure with large openings and a cavity inside. Substrates of E2 enter the active site from different directions, i.e. CoA come from inside the core, while lipoyl moieties come from the outside. Twelve monomers of BP (for eukaryotic PDCs) were suggested to bind within the cavity of E2 and interact with the E3 dimer inside 12 pentagonal openings. Yeast E2 core has the size of 250 Å with 40-Å variations in diameter as found by electron microscopy. The contraction and expansion of the E2 core was proposed to reflect the conformational mobility of PDC during catalysis [17]. Recently the structure of the whole PDC from bovine kidney was constructed based on cryoelectron microscopy (Fig. 4) [18]. Bovine PDC was found to have 60 monomers of E2, 12 monomers of BP, 22 tetramers of E1, and 6 dimers of E3. The size of the complex in the presence of E1 molecules is about 500 Å. E1 molecules are organized in trimers bound through the subunit-binding domains of E2 70 Å above the trimers of inner domains of E2. Lipoyl domains are proposed to rotate around the subunit-binding domains transferring intermediates between active sites of E1, E2, and E3, and this movement is provided by the conformational changes of the E2 core. E1 of PDC is a dimer of identical subunits in Gram-negative bacteria or a heterotetramer composed of 2 α and 2 β subunits in eukaryotes and Gram-positive bacteria. Both types of E1 have two active sites and require thiamine pyrophosphate (TPP) and Mg2+ as cofactors. Sequence similarity between the two types of E1 is low; however, there is a significant similarity in the structure of the TPP-binding region for several TPP-dependent enzymes, the three-dimensional structures of which are known (yeast transketolase, Lactobacillus plantarum pyruvate oxidase, yeast, and Zymomonas mobilis pyruvate decarboxylase, P. putida benzoylformate decarboxylase, human and P. putida branched-chain α-keto acid dehydrogenase, Desulfovibrio africanus pyruvate:ferredoxin oxidoreductase [19, 20], and E. coli PDC-E1 [15]). The common features of the active sites of these proteins are: (i) active sites are localized on the interface of two subunits; (ii) two residues of the TPP motif (a sequence of 30–33 amino acid residues conserved for all known TPP-requiring enzymes) coordinate binding of the pyrophosphate moiety of TPP through interactions with a divalent cation (Mg2+ or Ca2+); (iii) amino acid residues help TPP to maintain the “V” conformation necessary for catalysis (among them are phenylalanine or tyrosine residues providing a stacking interaction with the aminopyrimidine ring of TPP and a hydrophobic residue between two aminopyrimidine and thiazole rings); and (iv) N1′ of the pyrimidine ring binds to the glutamine residue through a hydrogen bond, which affects reactivity of the 4′-amino group of TPP and activates the C2 hydrogen of the thiazolium ring of TPP. Only the E. coli PDC-E1 structure has been The human PDC-E1 structure was The structure-function of E1 from human and E. coli have been studied E1 has two a form with the for on the and a form with the for on The second is necessary during the is or A. suum has two E1 in anaerobic and in E3 is a with two active sites localized the dimer active site tightly noncovalently bound which in the electron from dihydrolipoamide to NAD+ with of an active site subunit of E3 has central and interface Interestingly, E3, the product of the in different complexes, i.e. dehydrogenase branched-chain α-keto acid dehydrogenase and as in Only in P. putida three different for three dihydrolipoamide E3 is bound to the subunit-binding domain of BP in eukaryotic PDC and to the subunit-binding domain of E2 in other The structure of B. E3 bound to the subunit-binding domain of E2 was to The interaction between these proteins was to be i.e. between residues of the binding domain and residues of E3. The subunit-binding domain of E2, however, binds E1 the different E3 with the of the subunit-binding E1 binds to the of the The binding site for the A. E1 was to not only the subunit-binding domain also the E2 inner domain is a specific kinase that E1 of PDC and is present in plant, and has several in mammalian PDC and two in plants, and one in nematode and (Table a three specific residues site site and site based on the of of mammalian E1 of A. suum E1, and Only two sites are present in of A. suum E1 and in and only one site is in Yeast E1 has site in a sequence that can be by bovine however, is not in have low with eukaryotic protein and are to bacterial histidine protein Recently the three-dimensional structure of was The domain of to be similar to the domain of bacterial histidine However, catalyzes the directly to the residue of the and not through a histidine residue of the kinase as bacterial histidine and is to the eukaryotic and by catalytic is a dimer composed of identical were to bind the lipoyl domains of E2 and BP In mammalian PDC there are two lipoyl domains in E2 and and one in BP (Fig. of have to the lipoyl binds to and and can also bind and binds to and to The of molecules in the PDC is as low as one to three molecules complex. of from one lipoyl domain to is necessary to of E1 is bound to the lipoyl domain of E2 of is different for different of The be by the conformational changes of bound to the lipoyl domain and of and its E1 on E2 because by a lipoyl domain is with E2. E2 does not only bind and in PDC also the of through the state of the lipoyl is with the reduction and of the lipoyl of E2 during PDC reaction The is by an allosteric of a reduced or form of of the lipoyl domain in the The of on the with the found for is a composed of two the catalytic subunit to the phosphatase of and the regulatory which is a (Table I). of the catalytic subunit of are in mammalian tissues and with different and The regulatory subunit of affects the of the catalytic subunit to binds to the of E2 with a role The central role of PDC in glucose homeostasis the high of of its in mammalian of PDC within and will be to as a while a within to is as a The of PDC by and is through of E1 by to of E1 (and and and is accomplished by (Fig. 1). above of and two of the catalytic subunit of are present in mammalian of and have different specific to different and (Table [6, The of is present in and are present in and is in tissues with low in and is in and in and was in and with low in and high of is present in while of are higher in liver and adipose The presence of several sites of E1 to the of the of of PDC In mammalian PDC three sites are by different of all three and can only sites and 2 are bound to E1 in The of site are: the for site 2 are: In the absence of E2 are also The in the absence of E2 was site however, site 2 be by and site be by in the absence of E2. of each site in E1 The of of E1 by of three sites is of site interaction of the E1 active site with the of the pyruvate and with the lipoyl domain of E2, of site affects E1 interaction with TPP. The products of the PDC reaction (and the products of fatty acid and NADH and of through of lipoyl moieties of the lipoyl domains of E2 to which are the and the portion of PDC present in active The is for The is [6, of also on the as and are of and to a other are by the of the PDC reaction by pyruvate is with The of was suggested to be formation of the complex that and of are regulated by the of divalent Mg2+ and is a and changes in Mg2+ with the in the is to Mg2+ is of to Mg2+ changes it is bound to the regulatory subunit of the of both present and with a regulatory in conditions of energy of by its binding to E2. The of PDC is by which of in and of The from is by protein kinase that to it and activates have both mitochondrial and and only mitochondrial PDC is regulated by Interestingly, of PDC in on and a in mitochondrial PDC due to by the of ATP. In the of and Mg2+ in in during PDC conditions of are in the of have two of E1, present in anaerobic and in The two E1 have different of and sites in are to mammalian has only two sites that are to This with the reduced of nematode to the and in the PDC a high in anaerobic conditions in the absence of the tricarboxylic acid cycle the products of the PDC reaction are for fatty acid it is critical to have such a and of PDC function with sites as well as of and that are regulated by such a of This first in of on the available the are for such an of during the from the fed to fasting the needs to the (such as and from that can be for (Fig. The the complex by even only one of the three sites present in the α subunit of E1 α subunits present in E1 potential sites for and of one site is to E1 and are two possible for the sites of (i) the by of E1 by the during the conditions such as the from the to fed state and (ii) providing a to to maintain this one site is by the of the presence of of specific with in is linked to Since all tissues are not in in glucose the of in different tissues (such as in and in liver during fasting and in plays critical in the flux through Additionally, the of to the changes in the and due to in the oxidation of fatty acids (and in tissues an fasting not only up the action of specific also an the changes in such as NADH, and in the due to oxidation of fatty an for in changes in the of and in the that the of most the changes also the of PDC one of its component enzymes, the of is not to the complex; the changes in the through the allosteric of the of This of is linked to the of other in the of different the of the active state of PDC in the liver and are during the fed to fasting these tissues to fatty acids and complete oxidation of (Fig. In the of the active state of PDC in the brain is during the under dietary conditions the of the brain to glucose to CO2 and is not during an or of (Fig. This the brain to oxidation of glucose for energy However, the of are an of fasting or in the brain is to of the (such as acetyl-CoA and that PDC via on and glucose oxidation while energy homeostasis in the the and catalytic of PDC with the other two in the such as the dehydrogenase complex and the branched-chain α-keto acid dehydrogenase complex on the for or an absence of of these based on in the oxidative there is a complete of by phosphorylation/dephosphorylation of the dehydrogenase complex because of its central in of the tricarboxylic acid Since the tricarboxylic acid cycle is in the oxidation of all generated from the metabolism of carbohydrates, fats, and amino which are of the cycle for the of this complex not for in the In the of the branched-chain α-keto acid dehydrogenase complex by a phosphorylation/dephosphorylation does a for oxidation of the three amino acids and which a role in metabolism in the it is no that this complex is regulated by of one site only on its E1 component by a specific kinase and a specific phosphatase with only a of of this with the of of the branched-chain α-keto acid dehydrogenase complex is by a for its and reflect a specific role of this complex in the of metabolism for energy the of PDC by covalent modification is the key to the of glucose homeostasis during the between the fed and in PDC (and PDC in to dietary and changes are also PDC in liver and adipose by and in fed a high for with that of The of the component proteins of PDC by in from high with changes in PDC In PDC was in from high Interestingly, and active PDC were not in and of fed the high or high in the about reduction in PDC in the and this was with a similar reduction in of E1 proteins other components of PDC were not changes in PDC are by the for PDC components were to be regulated through different in Interestingly, of and in high glucose in PDC and this was with the of and E1β The glucose of and E1β in due to the presence of in the of the and E1β The major of the PDC under different is the of in the of in different conditions is well and studied the of the of is for and to a for However, the of in all tissues its the of and in mammary and and in and not the protein of is higher in adipose and is the only in which PDC does not during fasting because brain on glucose as a major of the of in and and the of in of the changes in In adipose PDC is regulated by which activates of or by of to in of protein in and and of protein in and a high are conditions to higher of fatty acids are known to α of is found to of in and liver and in the in Both without its on the other the of and It is proposed that of fatty acids in conditions of and through of The of and is suggested to be regulated to some by the in Recently of was to in and in of of and of to on of E1 and of of one of the E1 sites is first in the is for however, in a of of one site requires a for and of the of the three human PDC and and the have been have been found to the α subunit of the E1 component of PDC in most mammals. and both localized on are in human 4) and in the are in only are the of the and (and of (Fig. The presence of several as well as domains in the region of the human have been by sequence and of the region of the human that the sequence to as the sequence and a for the this as a it was that the site is and the sequence the to region was for the of the human acetyltransferase was to of in kidney and adipose not in the brain of However, this not sequence to regulated in several it was that the and of the regulatory that are to function in a In the region of the human was to be to glucose in of the human region the presence of two from to and from to that are for the in these two variations of the in other the sequence a large on It be that of human was relatively low with that of the kinase and the This be due to the of two of the sequence by an in the human In to of of the of in is tightly regulated that its only during specific of of a core region has revealed of of these potential for and protein and the two binding sites site 2) and a complex with a binding of the of human the presence of domains within the region and three and (Fig. also the presence of both and in the that are only in PDC is one of the major in of PDC have been with that are to the central due to the on glucose oxidation for energy that the human PDC is composed of several different a involving one of them to the in its of all of PDC of the E1 all of which are found in the α and the are among the other components not and the presence of with for of one only with in which there is some PDC can This is well by of the in in Since the human is localized on the of of in different and tissues in to the in PDC with In PDC the of PDC in glucose metabolism in the developing mammalian large of specific with the PDC components have been all of which are within the region Interestingly, about of from changes the structure and some catalytic function of the of these and based on of the residues in α subunit were by human E1 in E. coli to the for function Interestingly, of these the binding of TPP in the E1 active of have the of in and low or even in in a daily of large of thiamine TPP for some and of and the PDC in a active all of these have with and The central role of PDC in glucose homeostasis has been for several and has been in of as a for glucose metabolism during during the The flux through PDC is by an via phosphorylation/dephosphorylation that is not only to metabolic such as the of and also to the interactions among the component proteins of PDC such as E2, and that interactions in PDC to the the and of the regulatory and as in this the necessary for of PDC and its in the of glucose homeostasis in mammals. of the of PDC in both glucose and energy homeostasis not only in also from early does not in the complete absence of of PDC by phosphorylation/dephosphorylation by and residues of E1 sites are as and in and as and PDC lipoyl moiety of E2. The domains of E2 and of bovine PDC and and of the domains of E2 subunit of of symmetry of the three-dimensional of the bovine kidney PDC and with the to the that binds E1 to the E2 core The inner is Å in the domain is for the of the dodecahedral to which E1 and The of the E2 the and lipoyl the and The inner is revealed in the three-dimensional structure of the and are with from [18]. of A. in and glucose metabolism in mammalian and brain during the fed and states. The central role of PDC in glucose during the fed state and in glucose during the state is The flux of fatty acids and in the and brain are by and tricarboxylic acid low amino of specific and domains of the of the human and are to of the of of and of this for critical of the and