Α-Keto ACID DEHYDROGENASE COMPLEXES, X. REGULATION OF THE ACTIVITY OF THE PYRUVATE DEHYDROGENASE COMPLEX FROM BEEF KIDNEY MITOCHONDRIA BY PHOSPHORYLATION AND DEPHOSPHORYLATION

Abstract

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

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“…A prototype of such a loop is pyruvate dehydrogenase kinase-1 (PDK-1) which phosphorylates pyruvate dehydrogenase (PDH). PDH fuels the mitochondrial TCA cycle via conversion of pyruvate to the TCA cycle substrate acetyl-coenzyme A (ac-CoA) [25]. PDK-1 mediates inactivating phosphorylation of PDH shutting down the ac-CoA supply of the TCA cycle.…”

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“…The PDHC is comprised of several copies of enzymatic subunits, E1 (pyruvate dehydrogenase), E2 (dihydrolipoamide transacetylase) and E3 (dihydrolipoamide dehydrogenase) and an E3 binding protein (E3BP), and its activity is regulated by several isoforms of kinases and phosphatases through reversible phosphorylation (Linn et al 1969;Pagliarini and Dixon 2006). The E1 enzyme consists of two a-and two b-subunits that share a binding site for thiamine pyrophosphate (TPP), a metabolite of thiamine, which is an essential cofactor for the enzymatic reaction and also helps to maintain the PDHC in an activated state by inhibiting its phosphorylation (Roche and Reed 1972).…”

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“…Although PDK isozymes are bound to the acetyltransferase component, they phosphorylate the dehydrogenase components (E1) of the multienzyme complex (16). In PDC, there are approximately 20-30 copies of pyruvate dehydrogenase physically attached to the core made of 60 copies of E2 and 12 copies of E3BP (10).…”