The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In ␣ 2 ␤ 2 -heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the C ␣ ؊C(‫؍‬O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-Å resolution. We propose a model for the flip-flop action of this enzyme through a concerted ϳ2-Å shuttlelike motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.The thiamin pyrophosphate (TPP) 1 -dependent enzymes perform a wide range of catalytic functions in the pathways of energy production, including decarboxylation of ␣-keto acids followed by transketolation. The enzymes that have been structurally characterized so far, 2-oxoisovalerate dehydrogenase from Pseudomonas putida (1), human branched-chain ␣-ketoacid dehydrogenase (2), bacterial pyruvate dehydrogenase (3), transketolase (4), pyruvate decarboxylase (5), benzoylformate decarboxylase (6), acetohydroxyacid synthase (7), pyruvate oxidase (8), and pyruvate:ferredoxin oxidoreductase (9), have shown a common mechanism of TPP activation by (i) forming the ionic N-H⅐⅐⅐O Ϫ hydrogen bonding between the N1Ј atom of the aminopyrimidine ring of the coenzyme and an intrinsic ␥-carboxylate group of glutamate and (ii) imposing an "active" V-conformation that brings the N4Ј atom of the aminopyrimidine to the distance required for the intramolecular C-H⅐⅐⅐N hydrogen-bonding with the thiazolium C2 atom (Fig. 1). Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1Ј atom of the aminopyrimidine system is strictly connected with the deprotonation of the 4Ј-amino group in that system and eventually abstraction of the proton from C2 and formation of the reactive 4Ј-amino-C2-carbanion (Fig. 1a) (10). This reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used in the family of TPP-dependent enzymes. Within pyruvate dehydrogenase (E1), the first catalytic component enzyme of pyruvate dehydrogenase complex (PDC), this substrate is pyruvate (S 1 ). The cleavage of the central C ␣ -C(ϭO) bond of this substrate proceeds from induction of the intermediate, 4Ј-imino-2-(2-hydroxypropionyl)thiamin pyrophosphate, i.e. lactyl-TPP (LTPP) (Fig. 1b), followed by conversion to 4Ј-imino-2-(1-hydroxyethyl) thiamin pyrophosphate (HETPP) with release of carbon dioxide (P 1 ) (Fig. 1c). The fate of this active C2-␣-carbanion/enamine HETPP differs among various TPP-dependent enzymes depending on the nature of t...

At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residues in the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHc correlates mostly with phosphorylation of serine 264, which is located at the entrance of the substrate channel leading to the active site of E1. Despite intense investigations, the molecular mechanism of this inactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in human wild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation of Ser264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvate decarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active site via the substrate channel and the subsequent reductive acetylation of the E2 component are severely slowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Glu determined by X-ray crystallography reveals no differences in the three-dimensional architecture of the phosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However, the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant. By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glu suggest that phosphorylation blocks access to the active site by imposing a steric and electrostatic barrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant, which carries no charge at position 264, is also selectively deficient in pyruvate binding and reductive acetylation of E2, we conclude that mostly steric effects account for inhibition of PDHc by phosphorylation.

The human (h) pyruvate dehydrogenase complex (hPDC) consists of multiple copies of several components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3), E3-binding protein (BP), and specific kinases and phosphatases. Mammalian PDC has a well organized structure with an icosahedral symmetry of the central E2/BP core to which the other component proteins bind non-covalently. Both hE2 and hBP consist of three well defined domains, namely the lipoyl domain, the subunit-binding domain and the inner domain, connected with flexible linkers. hE1 (α2β2) binds to the subunit-binding domain of hE2; whereas hE3 binds to the E3-binding domain of hBP. Among several residues of the C-terminal surface of the hE1β E1βD289 was found to interact with hE2K276. The C-terminal residue I329 of the hE1β did not participate in binding to hE2. This latter finding shows specificity in the interaction between E1β and E2 in hPDC. The selective binding between hE3 and the E3-binding domain of hBP was investigated using specific mutants. E3R460G and E3340K showed significant reductions in affinity for hBP as determined by surface plasmon resonance. Both residues are involved in the structural organization of the binding site on hE3. Substitution of I157, N137 and R155 of hBP resulted in variable increases in the KD for binding with wild-type hE3, suggesting that the binding results from several weak electrostatic bonds and hydrophobic interactions among residues of hBP with residues at the interface of dimeric hE3. These results provide insight in the mono-specificity of binding of E1 to E2 and E3 to BP in hPDC and showed the differences in the binding of peripheral components (E1 and E3) in human and bacterial PDCs.

No abstract

MS.Tissue-specific pyruvate dehydrogenase complex deficiency causes cardiac hypertrophy and sudden death of weaned male mice. Am J Physiol Heart Circ Physiol 295: H946 -H952, 2008. First published June 27, 2008 doi:10.1152/ajpheart.00363.2008.-Pyruvate dehydrogenase complex (PDC) plays an important role in energy homeostasis in the heart by catalyzing the oxidative decarboxylation of pyruvate derived primarily from glucose and lactate. Because various pathophysiological states can markedly alter cardiac glucose metabolism and PDC has been shown to be altered in response to chronic ischemia, cardiac physiology of a mouse model with knockout of the ␣-subunit of the pyruvate dehydrogenase component of PDC in heart/skeletal muscle (H/SM-PDCKO) was investigated. H/SM-PDCKO mice did not show embryonic lethality and grew normally during the preweaning period. Heart and skeletal muscle of homozygous male mice had very low PDC activity (ϳ5% of wild-type), and PDC activity in these tissues from heterozygous females was ϳ50%. Male mice did not survive for Ͼ7 days after weaning on a rodent chow diet. However, they survived on a high-fat diet and developed left ventricular hypertrophy and reduced left ventricular systolic function compared with wild-type male mice. The changes in the heterozygote female mice were of lesser severity. The deficiency of PDC in H/SM-PDCKO male mice greatly compromises the ability of the heart to oxidize glucose for the generation of energy (and hence cardiac function) and results in cardiac pathological changes. This mouse model demonstrates the importance of glucose oxidation in cardiac energetics and function under basal conditions. Pdha1 gene deletion; ventricular hypertrophy; high fat diet; sudden death THE HEALTHY ADULT MAMMALIAN heart derives 60 -90% of ATP from fatty acid oxidation, 10 -40% from glucose and lactate oxidation in the tricarboxylic acid (TCA) cycle [through the pyruvate dehydrogenase (PDH) complex (PDC)], and Ͻ2% from glycolysis (9,21,22). The pathways of uptake and oxidation of fatty acids and glucose are tightly regulated because the heart has limited storage capacity for fatty acids and glucose, and it needs to respond to the changes in fuel availability and energy demands. The switch in the fuel selection provides for constant ATP production despite different developmental, dietary, and pathophysiological conditions. For example, the fetal heart relies more on glucose metabolism, whereas fatty acid oxidation is the primary energy source for the adult heart (15). Different pathological conditions such as cardiac hypertrophy, hypoxia, and ischemia change cardiac metabolism toward glucose utilization, whereas diabetes shifts metabolism toward fatty acid oxidation. Exercise and fasting conditions also lead to increases in fatty acid oxidation and decreases in glucose oxidation (9,21,22).PDC plays a key role in glucose metabolism by linking glycolysis and the TCA cycle. PDC is a highly organized multienzyme complex composed of three catalytic components (PDH, dihydrolipoamid...

The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.

Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the ␣ subunit of ␣ 2 ␤ 2 PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37°C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 ␣ subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. Mammalian pyruvate dehydrogenase complex (PDC)2 plays a central role in glucose oxidation by catalyzing the oxidative decarboxylation of pyruvic acid with formation of carbon dioxide, acetyl-CoA, NADH, and H ϩ (for reviews see Refs. 1-3). PDC is composed of multiple copies of the following three catalytic components: (i) pyruvate dehydrogenase (PDH) catalyzing the decarboxylation of pyruvate and reductive acetylation of the lipoyl moieties of dihydrolipoamide acetyltransferase (E2); (ii) E2 transferring acetyl moiety to CoA; (iii) and dihydrolipoamide dehydrogenase (E3) reoxidizing the reduced lipoyl moieties of E2 with the reduction of NAD ϩ to NADH (1-3). Forty eight to 60 subunits of E2and 12 subunits of E3-binding protein (BP), a noncatalytic component of mammalian PDC, form the central core of the multienzyme PDC, which binds 20 -30 tetramers of PDH, 6 -12 dimers of E3 and two regulatory enzymes, 1-2 copies of pyruvate dehydrogenase kinase (PDK), and 2-3 copies of pyruvate dehydrogenase phosphatase (PDP) (4, 5). PDK and PDP provide the finely tuned regulation of activity of PDC through reversible phosphorylation-dephosphorylation of PDH (and hence PDC) depending on cellular demand for glucose oxidation. Mammalian PDH is a tetramer consisting of two ␣ and two ␤ subunits. The hum...

No abstract