Lester J. Reed

α-Keto acid dehydrogenase complexes: XV. Purification and properties of the component enzymes of the pyruvate dehydrogenase complexes from bovine kidney and heart☆

Abstract Procedures are described for isolation of the pyruvate and α-ketoglutarate dehydrogenase complexes in good yield and in a highly purified state from bovine kidney and heart mitochondria. Procedures are presented for separation of the kidney and heart pyruvate dehydrogenase complexes into their component enzymes and for purification of the individual enzymes. The nonphosphorylated and phosphorylated forms of the pyruvate dehydrogenase component of both the kidney and heart complexes were crystallized. Pyruvate dehydrogenase kinase, a regulatory enzyme, is tightly bound to the dihydrolipoyl transacetylase component of the kidney and heart complexes. The kinase was separated and purified from the kidney transacetylase. A second regulatory enzyme, pyruvate dehydrogenase phosphatase, appears to be loosely associated with the kidney and heart pyruvate dehydrogenase complexes. The kidney and heart pyruvate dehydrogenase phosphatases were purified 400- to 1000-fold from mitochondrial extracts. The heart phosphatase is close to being homogeneous. The molecular weight of the two phosphatases was estimated by gel filtration to be about 100,000. Preliminary data indicate that the molar concentration of the kinase and the phosphatase is about an order of magnitude less than that of their protein substrate, pyruvate dehydrogenase.

[12] α-ketoglutarate dehydrogenase complex from Escherichia coli

Publisher Summary The chapter describes assay method, purification procedure, and properties of the α- ketoglutarate dehydrogenase enzyme. This enzyme has been isolated from pig heart muscle, Escherichia coli, and beef kidney mitochondria as multienzyme complexes with molecular weights of several million. The E. coli α-ketoglutarate dehydrogenase complex is separated in three enzymes, α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3), and the complex is reconstituted from the isolated enzymes. There is as no sensitive and convenient assay for the a-ketoglutarate dehydrogenase complex that may be used at all levels of purity of the enzyme complex. The DPN reduction assay described is the only reliable assay for the intact complex however it is not suitable for crude preparations that contain DPNH oxidase. Assays for the three component enzymes of the a-ketoglutarate dehydrogenase complex are described, but these assays do not distinguish between the free and combined enzymes. Thus, the ferricyanide reduction assay described provides an estimate of the activity of the α-ketoglutarate dehydrogenase component (E1) of the complex.

Regulation of pyruvate dehydrogenase kinase and phosphatase by acetyl-CoA/CoA and NADH/NAD ratios

Summary The interconversion of the active, nonphosphorylated form of pyruvate dehydrogenase and its inactive, phosphorylated form is modulated by acetyl-CoA/CoA and NADH/NAD molar ratios. An increase in either ratio increases the proportion of the phosphorylated form of pyruvate dehydrogenase. The activity of pyruvate dehydrogenase kinase is stimulated by acetyl-CoA and by NADH and is inhibited by CoA and by NAD. NADH inhibits pyruvate dehydrogenase phosphatase, and this inhibition is reversed by NAD.

A Trail of Research from Lipoic Acid to α-Keto Acid Dehydrogenase Complexes

In this article I shall retrace a trail of research that began with the isolation and characterization of a microbial growth factor and led to elucidation of the structure, function, and regulation of -keto acid dehydrogenase complexes. The high points of this trail are presented below. Isolation and Characterization of Lipoic Acid This trail of discovery started in the spring of 1949, about 6 months after I joined the faculty of the Department of Chemistry at the University of Texas. At that time I started working on the isolation of a factor that replaced acetate in the growth medium for certain lactic acid bacteria. Research on the “acetate-replacing factor” was initiated by Esmond Snell and associates at the University of Wisconsin and then at the University of Texas. I inherited this project in the spring of 1949. We established that this factor is widely distributed in animal, plant, and microbial cells and that liver is a rich source. The factor is tightly bound to liver protein and is released by proteolysis or by acid hydrolysis. At that time pharmaceutical companies were processing large amounts of pork and beef liver to obtain extracts suitable for treatment of pernicious anemia. The active principle was shown later to be vitamin B12. Fresh liver was extracted with warm water, and the residual liver proteins and fatty material were dried and sold as an animal feed supplement. Arrangements were made with Eli Lilly and Co. to obtain liver residue, and we developed procedures for extracting and purifying the acetatereplacing factor. We progressed to the point of being able to process about 6 pounds of liver residue at a time. A 16,000–50,000-fold purification was achieved. In the late 1940s and early 1950s several other groups were trying to isolate factors that were similar to, if not identical with, the acetate-replacing factor. These factors included the “pyruvate oxidation factor” of I. C. Gunsalus and associates that was necessary for oxidation of pyruvate to acetate and carbon dioxide by Streptococcus faecalis cells grown in a synthetic medium. Gunsalus was also collaborating with Eli Lilly and Co. In the fall of 1950, the Lilly Research Laboratories merged the two separate collaborations to facilitate isolation of the acetate-replacing/pyruvate oxidation factor. The Lilly group adapted and scaled up isolation procedures developed by us. Instead of processing 6-pound batches of liver residue at a time, they were able (using commercial equipment) to process 250-pound batches. Concentrates of the factor that were 0.1–1% pure were sent to my laboratory for further processing. I obtained the first pale yellow crystals of the factor, about 3 mg, on or about March 15, 1951, a truly memorable occasion. It was partially characterized and given the trivial name -lipoic acid (1). The isolation involved a 300,000-fold purification. A total of 30 mg of crystalline lipoic acid was eventually isolated. We estimated that 10 tons of liver residue were processed to obtain this small amount of the pure substance. And to think that I was processing about 6 pounds of liver residue at a time, convinced that I would eventually isolate the pure material. We established that lipoic acid is a cyclic disulfide, either 6,8-, 5,8-, or 4,8-dithiooctanoic acid. That the correct structure is 6,8-dithiooctanoic acid (1,2-dithiolane-3-valeric acid) was established by synthesis of DL-lipoic acid, first achieved by E. L. R. Stokstad and associates at Lederle THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 42, Issue of October 19, pp. 38329–38336, 2001

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 .

The remarkable structural and functional organization of the eukaryotic pyruvate dehydrogenase complexes

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (Mr ≈ 7.8 × 106) comprising about 22 molecules of pyruvate dehydrogenase (E1) and about 6 molecules of dihydrolipoamide dehydrogenase (E3) with its binding protein associated with the 60-subunit dihydrolipoamide acetyltransferase (E2) core provides considerable insight into the structural and functional organization of the largest multienzyme complex known. The structure shows that potentially 60 centers for acetyl-CoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E1 molecules bound to one E2 trimer adjacent to an E3 molecule in each of 12 pentagonal openings. The E1 components are anchored to the E1-binding domain of the E2 subunits through an ≈50-Å-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E2 trimer and form a cage around its base that may shelter the lipoyl domains and the E1 and E2 active sites. The docking of the atomic structures of E1 and the E1 binding and lipoyl domains of E2 in the electron microscopy map gives a good fit and indicates that the E1 active site is ≈95 Å above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E1-binding domain of E2, which is centrally located 45–50 Å from the E1, E2, and E3 active sites, and that the highly flexible breathing core augments the transfer of intermediates between active sites.

α-Keto acid dehydrogenase complexes: XVI. Studies on the subunit structure of the pyruvate dehydrogenase complexes from bovine kidney and heart☆

Abstract The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (a flavoprotein) are joined. Electron microscopic studies indicated that the design of the transacetylase is based on icosahedral (532) symmetry and, therefore, it would be expected to contain 60 very similar, if not identical, polypeptide chains. Evidence is presented that the bovine kidney and heart dihydrolipoyl transacetylases are very similar and that each enzyme does indeed consist of 60 apparently identical polypeptide chains. Each chain apparently contains 1 molecule of covalently bound lipoic acid. The molecular weight of the transacetylase is about 3.12 ± 0.20 million. The pyruvate dehydrogenase components of the bovine kidney and heart pyruvate dehydrogenase complexes are also very similar. The uncomplexed enzymes have a molecular weight of about 154,000 and possess the subunit structure α2β2. The molecular weights of two subunits are about 41,000 and 36,000, respectively. Only the α-subunit undergoes phosphorylation in the presence of pyruvate dehydrogenase kinase and ATP. Possible functions of the two subunits are discussed. It appears that the bovine kidney and heart pyruvate dehydrogenase complexes contain about 60 pyruvate dehydrogenase units (αβ) but only 10–12 flavoprotein chains.

[50] Purification and resolution of the pyruvate dehydrogenase complex (Escherichia coli)

Publisher Summary This chapter discusses the determination of purification and properties of the pyruvate dehydrogenase complex. The Escherichia coli pyruvate dehydrogenase complex has been separated into three enzymes, pyruvate decarboxylase (E 1 ), lipoyl reductasetransacetylase (E 2 ), and dihydrolipoyl dehydrogenase (E 3 ), and has been reconstituted from the isolated enzymes. In the case of dihydrolipoyl dehydrogenase, the assay is based on spectrophotometric determination of the rate of reduced diphosphopyridine nucleotide (DPNH) oxidation (at 340 mμ) in the presence of the dehydrogenase and lipoamide. In the case of pyruvate decarboxylase, the assay is a modification of that described by Hager. It is based on colorimetric determination of ferrocyanide (as Prussian blue) produced by oxidative decarboxylation of pyruvate with ferricyanide as electron acceptor. In the case of lipoyl reductase-transacetylase, the assay is based on colorimetric determination of S -acetyldihydrolipoamide as the ferric acethydroxamate complex. Acetyl CoA is generated from acetyl phosphate in the presence of catalytic amounts of CoA and phosphotransacetylase, and the acetyl group is transferred to dihydrolipoamide in the presence of lipoyl reductase-transacetylase.

Regulation of Mammalian Pyruvate and Branched-Chain α-Keto Acid Dehydrogenase Complexes by Phosphorylation — Dephosphorylation

Publisher Summary The pyruvate dehydrogenase multienzyme complex and the branched-chain α-keto acid dehydrogenase multienzyme complex are located in mitochondria, within the inner membrane matrix compartment. The pyruvate dehydrogenase multienzyme complex and the branched-chain α-keto acid dehydrogenase multienzyme complex are located in mitochondria within the inner membrane matrix compartment. The pyruvate dehydrogenase complex is well designed for fine regulation of its activity. Interconversion of the active and inactive forms of pyruvate dehydrogenase is a dynamic process that leads rapidly to the establishment of steady states, in which the fraction of phosphorylated enzyme can be varied progressively over a wide range by changing the concentration or molar ratios of effectors that regulate activities of the kinase and the phosphatase. The pyruvate dehydrogenase complex, like other interconvertible enzyme systems, functions uniquely as a metabolic integration system. By means of multisite interactions with allosteric effectors, the kinase and the phosphatase can sense simultaneous fluctuations in the intracellular concentrations of several metabolites and adjust the specific activity of pyruvate dehydrogenase accordingly. This chapter illustrates the phosphorylation and concomitant inactivation of the α-ketoisovalerate dehydrogenase complex by its endogenous kinase, and the dephosphorylation and reactivation by the addition of highly purified α-ketoisovalerate dehydrogenase phosphatase.

α-Keto acid dehydrogenase complexes: XVIII. Subunit composition of the Escherichia coli pyruvate dehydrogenase complex☆

Abstract The Escherichia coli Crookes pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (a flavoprotein) are joined by noncovalent bonds. Electron microscopic studies indicated that the design of the transacetylase is based on octahedral (432) symmetry and, therefore, it would be expected to contain 24 very similar, if not identical, polypeptide chains. Evidence is presented that the transacetylase does indeed consist of 24 apparently identical polypeptide chains. Each dehydrogenase (about 112,000). Both the uncomplexed pyruvate dehydrogenase and the flavoprotein contain two apparently identical polypeptide chains. The available data do not permit a decision as to whether each of these enzymes is present in the complex as a monomer or a dimer. It appears that the E. coli Crookes pyruvate dehydrogenase complex contains 24 pyruvate dehydrogenase chains, 24 dihydrolipoyl transacetylase chains, and 12 flavoprotein chains. Possible distributions of the pyruvate dehydrogenase chains and the flavoprotein chains around the transacetylase core are discussed.