Enzymes are biological catalysts that, like all catalysts, speed the approach to chemical equilibrium without shifting the equilibrium position itself. All

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and Summary.-This paper reports the discovery that the activity of the multienzyme pyruvate dehydrogenase complex from beef kidney mitochondria is regulated by a phosphorylation-dephosphorylation reaction sequence. The site of this regulation is the pyruvate dehydrogenase component of the complex. Phosphorylation and concomitant inactivation of pyruvate dehydrogenase are catalyzed by an ATP-specific kinase (i.e., a pyruvate dehydrogenase kinase), and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase (i.e., a pyruvate dehydrogenase phosphatase). The kinase and the phosphatase appear to be regulatory subunits of the pyruvate dehydrogenase complex.The pyruvate dehydrogenase complex (PDC) catalyzes a coordinated sequence of reactions that can be represented by over-all reaction (1). CH3COCO2H + CoA-SH + DPN + CH3CO-S-CoA + C02 + DPNH + H+.(1) This paper reports the discovery that the activity of PDC from beef kidney mitochondria is subject to regulation by a phosphorylation-dephosphorylation reaction sequence. The highly purified PDC is inactivated by incubation with low concentrations of ATP, and the inactive preparations are reactivated by incubation with 10 mM Mg++. Inactivation involves phosphorylation of the pyruvate dehydrogenase (PDH) component of the complex, and reactivation accompanies dephosphorylation. The phosphorylation and dephosphorylation reactions are catalyzed, respectively, by a kinase and a phosphatase which appear to be associated closely with or integral parts of the PDH. The significance of these findings with respect to regulation of pyruvate metabolism in kidney mitochondria is discussed below.Materials and Methods.-a_-P32-ATP and y-P'2-ATP were obtained from International Chemical and Nuclear Corp.; ,B, y-P'2-ATP came from Schwartz BioResearch; protamine sulfate was from Eli Lilly and Co.; and Sepharose 4B came from Pharmacia Fine Chemicals. The DPN-reduction assay for the intact PDC, which is based on reaction (1), and other assays were carried out as described previously.2 3 Protein-bound radioactivity was determined by precipitation of the radioactive protein on filter-paper disks. 4 Purification of PDC and KGDC: Beef kidney mitochondria were isolated in 0.25 M sucrose, washed with 20 mM phosphate buffer (pH 7.0 and 6.3), and frozen and thawed essentially as described in a previous publication.5 The thawed suspension was made 50 mM with respect to NaCl and was clarified by centrifugation. The mitochondrial ex-234

The highly purfied pyruvate dehydrogenase complex (EC 1.2.4.1) and uncomplexed pyruvate dehydrogenase from bovine kidney and heart mitochondria were phosphorylated and inactivated with pyruvate dehydrogenase kinase and [gamma-32P]ATP. Tryptic digestion of the phosphorylated pyruvate dehydrogenase yielded three phosphopeptides, a mono- (site 1) and a di- (sites 1 and 2) phosphorylated tetradecapeptide and a monophosphorylated nonapeptide (site 3). The amino acid sequences of the three phosphopeptides were established to be Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser-Tyr-Arg, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser(P)-Tyr-Arg, and Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg. Phosphorylation proceeded markedly faster at site 1 than at sites 2 and 3, and phosphorylation at site 1 correlated closely with inactivation of pyruvate dehydrogenase. Complete inactivation of pyruvate dehydrogenase was associated with incorporation at site 1 of 1.0--1.6 mol of phosphoryl groups per mol of enzyme. Since pyruvate dehydrogenase is a tetramer (alpha2beta2) and since phosphorylation occurs only on the alpha subunit, the possibility of half-site reactivity is considered.

The three-dimensional reconstruction of the bovine kidney pyruvate dehydrogenase complex (M r Ϸ 7.8 ؋ 10 6 ) comprising about 22 molecules of pyruvate dehydrogenase (E 1) and about 6 molecules of dihydrolipoamide dehydrogenase (E 3) 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 acetylCoA synthesis are organized in sets of three at each of the 20 vertices of the pentagonal dodecahedral core. These centers consist of three E 1 molecules bound to one E2 trimer adjacent to an E3 molecule in each of 12 pentagonal openings. The E1 components are anchored to the E 1-binding domain of the E2 subunits through an Ϸ50-Å-long linker. Three of these linkers emanate from the outside edges of the triangular base of the E 2 trimer and form a cage around its base that may shelter the lipoyl domains and the E 1 and E2 active sites. The docking of the atomic structures of E1 and the E 1 binding and lipoyl domains of E2 in the electron microscopy map gives a good fit and indicates that the E 1 active site is Ϸ95 Å above the base of the trimer. We propose that the lipoyl domains and its tether (swinging arm) rotate about the E 1-binding domain of E 2, 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. T he pyruvate dehydrogenase complex (PDC) serves as the link between glycolysis and the tricarboxylic acid cycle and generally has prominence in the description of these metabolic pathways because they serve as a major source of cellular energy. A central feature of PDCs is a 24-mer (Escherichia coli) or 60-mer (eukaryotes and some Gram-positive bacteria) core with the morphologies of a cube or pentagonal dodecahedron, respectively (1-4). The structures with the latter morphology comprise the largest (M r Ϸ 10 7 ) multienzyme complexes known. Even more remarkable than their exceptional size and morphology, these complexes encompass some of the most unusual features found in structural biology as described below.The E 2 core comprises the dihydrolipoamide acetyltransferase activity and is the only oligomeric enzyme complex known to be organized with the shape of a pentagonal dodecahedron. Moreover, the 250-Å-diameter dodecahedron has a very unusual feature: the tightly bound trimers at each of its 20 vertices seem to be interconnected by 30 flexible bridges enabling the core to ''breathe,'' as evidenced by an extraordinary size variability of 40 Å (17%) at room temperature. The breathing core apparently is a common feature in the phylogeny of the PDCs, suggesting that protein dynamics is an integral component of the function of these multienzyme complexes (5). Moreover, dodecahedral morphology of the core favors a synchronous or harmonious change in the length of the bridges that is relate...

A branched chain α-keto acid dehydrogenase-dihydrolipoyl transacylase complex was purified to apparent homogeneity from bovine kidney mitochondria. As usually isolated, the complex ( s 20, w = 40 S) contained little, if any, dihydrolipoyl dehydrogenase. When saturated with the latter enzyme the complex had a specific activity of about 12 μmol of α-ketoisovalerate oxidized per min per mg of protein at 30° with NAD + as electron acceptor. In addition to α-ketoisovalerate, the complex also oxidized α-ketoisocaproate, α-keto-β-methylvalerate, α-ketobutyrate, and pyruvate. The ratios of the specific activities were 2.0:1.5:1.0:1.0:0.4, and the apparent K m values were 40, 50, 37, 56, and 1000 μM. The complex was separated into its component enzymes. The branched chain α-keto acid dehydrogenase (6 S) consists of two different subunits with estimated molecular weights of 46,000 and 35,000. The dihydrolipoyl transacylase (20 S) contains apparently identical subunits of molecular weight about 52,000. In the electron microscope, the transacylase has the appearance of a cube, and the molecules of branched chain α-keto acid dehydrogenase appear to be distributed on the surface of the cube. In contrast to the pyruvate dehydrogenase complex of bovine kidney, the branched chain α-keto acid dehydrogenase complex apparently is not regulated by phosphorylation-dephosphorylation. Its activity, however, is subject to modulation by end-product inhibition.

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