Thomas E. Roche

Molecular biology and biochemistry of pyruvate dehydrogenase complexes.

In most organisms, the pyruvate dehydrogenase complex catalyzes the pivotal irreversible reaction that leads to the consumption of glucose in the aerobic, energy‐generating pathways. A combination of biochemical and molecular biology studies have greatly expanded our understanding of the overall structural organization of this multicomponent system, delineated the locations and elucidated the functions of structural domains of the catalytic components, and revealed significant evolutionary changes. Important to this progress was the deduction of the primary amino acid sequences from cDNA clones for each of the catalytic components from several species. The greatest detail is available for the FAD‐containing dihydrolipoamide dehydrogenase component, which is the only component for which tertiary structure information has recently emerged. For the dihydrolipoamide acetyltransferase core component, a similar but species‐variable multidomain structure is established that is responsible for the distinct architectures of the inner cores, the peripheral binding of the other components, and the conveyance of reaction intermediates between distantly separated active sites. A second lipoyl‐bearing component, protein X, has been shown to play a critical role in the organization and function of the complex from many higher organisms. Although much is known about the means of effector modulation of mammalian complex activity, identification of the signal eliciting its regulation by insulin still poses an exciting challenge.— Patel, M. S.; Roche, T. E. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J. 4: 3224‐3233; 1990.

α-Keto acid dehydrogenase complexes: XVII. Kinetic and regulatory properties of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase from bovine kidney and heart

Abstract Both pyruvate dehydrogenase (PDH) kinase and PDH phosphatase require a divalent cation for activity. Either Mg2+ or Mn2+, but not Ca2+, can satisfy this requirement. The apparent Km of the kinase for MgATP2− is about 0.02 m m , whereas the apparent Km of the phosphatase for Mg2+ is about 2 m m . The apparent Km of the phosphatase for Mn2+ is about 0.5 m m . The bovine kidney and heart PDH kinases exhibit pH optima about 7.0–7.2 in the presence of either Mg2+ or Mn2+. The pH optima of the corresponding PDH phosphatases are about 6.7–7.1 in the presence of Mg2+ and 7.5–7.6 in the presence of Mn2+. ADP is a competitive inhibitor of ATP. The apparent Ki for ADP is about 0.1 m m . No effect of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) on either the kinase or the phosphatase was observed in these studies. Inhibition of PDH phosphatase by fluoride ion, inorganic orthophosphate, EDTA and EGTA is described. Pyruvate protects the pyruvate dehydrogenase complex against inactivation by ATP, and this effect appears to be more pronounced with the bovine heart complex than with the bovine kidney complex. It appears that pyruvate exerts its inhibitory effect primarily on the PDH kinase. We have observed that the kidney kinase catalyzes a phosphorylation of casein. This reaction is insensitive to cyclic AMP, and it is inhibited by pyruvate and α-ketobutyrate, but not by α-ketoglutarate. Dihydrolipoyl transacetylase markedly stimulates the rate of phosphorylation of PDH by PDH kinase. We have found that the transacetylase lowers the apparent Km of the kinase for PDH from about 20 μ m to about 0.6 μ m .

Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms.

The mammalian pyruvate dehydrogenase complex (PDC) plays central and strategic roles in the control of the use of glucose-linked substrates as sources of oxidative energy or as precursors in the biosynthesis of fatty acids. The activity of this mitochondrial complex is regulated by the continuous operation of competing pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) reactions. The resulting interconversion cycle determines the fraction of active (nonphosphorylated) pyruvate dehydrogenase (E1) component. Tissue-specific and metabolic state-specific control is achieved by the selective expression and distinct regulatory properties of at least four PDK isozymes and two PDP isozymes. The PDK isoforms are members of a family of serine kinases that are not structurally related to cytoplasmic Ser/Thr/Tyr kinases. The catalytic subunits of the PDP isoforms are Mg2+-dependent members of the phosphatase 2C family that has binuclear metal-binding sites within the active site. The dihydrolipoyl acetyltransferase (E2) and the dihydrolipoyl dehydrogenase-binding protein (E3BP) are multidomain proteins that form the oligomeric core of the complex. One or more of their three lipoyl domains (two in E2) selectively bind each PDK and PDP1. These adaptive interactions predominantly influence the catalytic efficiencies and effector control of these regulatory enzymes. When fatty acids are the preferred source of acetyl-CoA and NADH, feedback inactivation of PDC is accomplished by the activity of certain kinase isoforms being stimulated upon preferentially binding a lipoyl domain containing a reductively acetylated lipoyl group. PDC activity is increased in Ca2+-sensitive tissues by elevating PDP1 activity via the Ca2+-dependent binding of PDP1 to a lipoyl domain of E2. During starvation, the irrecoverable loss of glucose carbons is restricted by minimizing PDC activity due to high kinase activity that results from the overexpression of specific kinase isoforms. Overexpression of the same PDK isoforms deleteriously hinders glucose consumption in unregulated diabetes.

Pyruvate dehydrogenase kinase regulatory mechanisms and inhibition in treating diabetes, heart ischemia, and cancer

Abstract.The fraction of pyruvate dehydrogenase complex (PDC) in the active form is reduced by the activities of dedicated PD kinase isozymes (PDK1, PDK2, PDK3 and PDK4). Via binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2 60mer), PDK rapidly access their E2-bound PD substrate. The E2-enhanced activity of the widely distributed PDK2 is limited by dissociation of ADP from its C-terminal catalytic domain, and this is further slowed by pyruvate binding to the N-terminal regulatory (R) domain. Via the reverse of the PDC reaction, NADH and acetyl-CoA reductively acetylate lipoyl group of L2, which binds to the R domain and stimulates PDK2 activity by speeding up ADP dissociation. Activation of PDC by synthetic PDK inhibitors binding at the pyruvate or lipoyl binding sites decreased damage during heart ischemia and lowered blood glucose in insulin-resistant animals. PDC activation also triggers apoptosis in cancer cells that selectively convert glucose to lactate.

Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions

A highly sensitive spectrophotometric assay was developed for measuring flavin-containing monooxygenase activity using methimazole (N-methyl-2-mercaptoimidazole) as the substrate. With the procedure described, flavin-containing monooxygenase activity can be accurately measured in whole cell homogenates without interference due to NADPH oxidase activities. The effects of detergents and octylamine on female mouse liver flavin-containing monooxygenase activity were characterized for whole homogenates and microsomes prepared under conditions which tend to cause or minimize microsomal aggregation. A small activation was observed with 0.2% (v/v) Emulgen 913 with nonaggregated microsomes; higher levels of detergents gave maximal activity with aggregated microsomes. Variations in the activity of the female mouse liver enzyme with nutritional state and time of day were evaluated. Higher specific activities were observed in homogenates and microsomes of livers from fed animals than from livers of 24-h starved animals, and higher specific activities were present in samples from livers of animals sacrificed in late afternoon than in the early morning. In the period where activity increased in fed animals (i.e., the AM to PM transition), a portion of flavin-containing monooxygenase was more resistant to thermal inactivation. Other properties are described which suggest structural differences for at least a portion of the flavin-containing monooxygenase. The possibility that these differences may be related to turnover of the flavin-containing monooxygenase is discussed.

Alpha-keto acid dehydrogenase complexes

Interaction of protein domains in the assembly and mechanism of 2-oxo acid dehydrogenase multienzyme complexes.- Probing the active site of mammalian pyruvate dehydrogenase.- Role of the E2 core in the dominant mechanisms of regulatory control of mammalian pyruvate dehydrogenase complex.- Lipoamide dehydrogenase.- Plant pyruvate dehydrogenase complexes.- Regulation of the pyruvate dehydrogenase complex during the aerobic/anaerobic transition in the development of the parasitic nematode, Ascaris suum.- Structure, function and assembly of mammalian branched-chain ?-ketoacid dehydrogenase complex.- Lipoylation of E2 component.- Pyruvate dehydrogenase phosphatase.- The mitochondrial ?-ketoacid dehydrogenase kinases: Molecular cloning, tissue-specific expression and primary structure analysis.- Shorter term and longer term regulation of pyruvate dehydrogenase kinases.- Hormonal and nutritional modulation of PDHC activity status.- Regulation of branched-chain ?-keto acid dehydrogenase complex in rat liver and skeletal muscle by exercise and nutrition.- Dephosphorylation of PDH by phosphoprotein phosphatases and its allosteric regulation by inositol glycans.- Long-term regulation and promoter analysis of mammalian pyruvate dehydrogenase complex.- The sperm-specific pyruvate dehydrogenase E1? genes.- Molecular defects of the branched-chain ?-keto acid dehydrogenase complex: maple syrup urine disease due to mutations of the E1? or E1? subunit gene.- Human defects of the pyruvate dehydrogenase complex.- Multigenic basis for maple syrup urine disease with emphasis on mutations in branched chain dihydrolipoyl acyltransferase.- Structure and chromosomal localization of the human 2-oxoglutarate dehydrogenase gene.- Pyruvate dehydrogenase complex as an autoantigen in primary biliary cirrhosis.

Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 μm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k catalong with about a 2-fold decrease in the K m of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g.using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2–60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated ≥3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Abstract Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10 6 for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K i to K m ratio for NADH versus NAD + and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD + and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K + and NH + 4 decreased the K m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.

Lipoyl Domain-based Mechanism for the Integrated Feedback Control of the Pyruvate Dehydrogenase Complex by Enhancement of Pyruvate Dehydrogenase Kinase Activity

To conserve carbohydrate reserves, the reaction of the pyruvate dehydrogenase complex (PDC) must be down-regulated when the citric acid cycle is provided sufficient acetyl-CoA. PDC activity is reduced primarily through increased phosphorylation of its pyruvate dehydrogenase (E1) component due to E1 kinase activity being markedly enhanced by elevated intramitochondrial NADH:NAD+ and acetyl-CoA:CoA ratios. A mechanism is evaluated in which enhanced kinase activity is facilitated by the build-up of the reduced and acetylated forms of the lipoyl moieties of the dihydrolipoyl acetyltransferase (E2) component through using NADH and acetyl-CoA in the reverse of the downstream reactions of the complex. Using a peptide substrate, kinase activity was stimulated by these products, ruling out the possibility kinase activity is increased due to changes in the reaction state of its substrate, E1 (thiamin pyrophosphate). Each E2 subunit contains two lipoyl domains, an NH2-terminal (L1) and the inward lipoyl domain (L2), which were individually produced in fully lipoylated forms by recombinant techniques. Although reduction and acetylation of the L1 domain or free lipoamide increased kinase activity, those modifications of the lipoate of the kinase-binding L2 domain gave much greater enhancements of kinase activity. The large stimulation of the kinase generated by acetyl-CoA only occurred upon addition of the transacetylase-catalyzing (lipoyl domain-free) inner core portion of E2 plus a reduced lipoate source, affirming that acetylation of this prosthetic group is an essential mechanistic step for acetyl-CoA enhancing kinase activity. Similarly, the lesser stimulation of kinase activity by just NADH required a lipoate source, supporting the need for lipoate reduction by E3 catalysis. Complete enzymatic delipoylation of PDC, the E2-kinase subcomplex, or recombinant L2 abolished the stimulatory effects of NADH and acetyl-CoA. Retention of a small portion of PDC lipoates lowered kinase activity but allowed stimulation of this residual kinase activity by these products. Reintroduction of lipoyl moieties, using lipoyl protein ligase, restored the capacity of the E2 core to support high kinase activity along with stimulation of that activity up to 3-fold by NADH and acetyl-CoA. As suggested by those results, the enhancement of kinase activity is very responsive to reductive acetylation with a half-maximal stimulation achieved with ∼20% of free L2 acetylated and, from an analysis of previous results, with acetylation of only 3-6 of the 60 L2 domains in intact PDC. Based on these findings, we suggest that kinase stimulation results from modification of the lipoate of an L2 domain that becomes specifically engaged in binding the kinase. In conclusion, kinase activity is attenuated through a substantial range in response to modest changes in the proportion of oxidized, reduced, and acetylated lipoyl moieties of the L2 domain of E2 produced by fluctuations in the NADH:NAD+ and acetyl-CoA:CoA ratios as translated by the rapid and reversible E3 and E2 reactions.

Function of the nonidentical subunits of mammalian pyruvate dehydrogenase

Abstract The pyruvate dehydrogenase (PDH) component of the bovine kidney pyruvate dehydrogenase complex (PDC) contains two nonidentical subunits. PDH catalyzes the decarboxylation of pyruvate to produce α-hydroxyethylthiamine-PP (HETPP) and the reductive acetylation of the lipoyl moieties of dihydrolipoyl transacetylase with HETPP. Phosphorylation of PDH with PDH kinase and ATP markedly inhibits the first reaction but does not inhibit the second reaction. Since the α-subunit but not the β-subunit of PDH undergoes phosphorylation, these results suggest that the α-subunit catalyzes the first reaction and the β-subunit catalyzes the second reaction. Thiamine-PP reduces the rate of phosphorylation of PDC by PDH kinase and ATP. Phosphorylation of PDC increases the KD of the PDC-Mg-thiamine-PP complex about 12-fold. It appears that the thiamine-PP binding site and the phosphorylation site on PDH influence each other and that HETPP is bound to PDH in a different orientation or possibly at a different site than is thiamine-PP.