Chemical modification of the essential arginine residues of pyruvate dehydrogenase prevents its phosphorylation by kinase

Nemerya, N.S.; Zemskova, Marina; Nyukhalkina, I.A.; Khailova, Ljudmila S.

doi:10.1016/0014-5793(96)00871-x

Cited by 5 publications

(5 citation statements)

References 13 publications

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“…The partial protection from phosphorylation (inactivation) of the enzyme provided by this analog does not rule out that the phosphorylation and substrate binding sites are close to each other. Competition for the positively charged guanidinium group of the arginine residue, which binds the pyruvate carboxylate [16], may block substrate binding to the active sites of phosphorylated El. …”

Section: Discussionmentioning

confidence: 99%

The effect of phosphorylation on pyruvate dehydrogenase

1995

FEBS Letters

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Phosphorylation of the pyruvate dehydrogenase component (El) of the muscle pyrnvate dehydrogenase complex (PDC) by El-kinase inhibits substrate conversion both in oxidative and non-oxidative reactions. Circular dichroism spectra were used to monitor the effect of phosphorylation on the following stages of the process: holoform formation from apo-E1 and thiamine pyrophosphate (TPP), substrate binding and active site deacetylation. It has been shown that phosphorylation of E1 reduces its affinity for TPP and prevents holo-E1 interaction with pyruvate. Phosphorylated and dephosphorylated PDC convert 2-hydroxyethyl-TPP in similar ways involving half of their active sites; all active sites of E1 function in the presence of deacetylating agents. The data obtained suggest that the pbosphorylation and substrate binding sites interact with each other.Key words': Pyruvate dehydrogenase; Protein kinase; Phosphorylation; Circular dichroism ExperimentalPDC was isolated from pigeon breast muscle according to Jagannathan and Schweet [10], with some modifications. The PDC activity was estimated from the rate of NADH production in the presence of CoA [11]. E 1 activity was determined with an artificial electron acceptor (2,6-dichlorophenolindophenol) as described previously [12]. Pyruvate consumption due to non-oxidative action of PDC was measured as follows: PDC (native or phosphorylated by endogenous E l-kinase with ATP) was incubated in the presence of excess TPP and pyruvate. Aliquotes were taken in time and the residual level of the substrate was determined from NADH decrease in the presence of lactate dehydrogenase. El-kinase was assayed by following PDC inactivation in the presence of ATE The incubation medium contained 0.1 M potassium phosphate buffer, pH 7.0, 0.5-2 mg/ml PDC, 0.1 M MgC12 and varied amounts of ATE Aliquots were withdrawn to be assayed for the enzyme activity. Circular dichroism (CD) spectra were recorded on a CNRS-Russel-Jouan Dichrograph 111 as described earlier [7,9]. The data obtained were expressed in the form of the ratio Ae/c, where Ae is the differential dichroic absorbance, expressed in optical scale units; c is the protein concentration in mg/ml. Acetoin was assayed by the method of Westerfeld [13].

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Section: Discussionmentioning

confidence: 99%

The effect of phosphorylation on pyruvate dehydrogenase

1995

FEBS Letters

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“…The studies on the mechanism of inactivation of mammalian and avian E1s showed the effect of E1 phosphorylation on the affinity for TPP and the formation of enzyme-bound HE=TPP from E1-TPP in either the forward (pyruvate as a substrate) or reverse reaction (acetylhydrolipoate as a substrate) (Roche and Reed, 1972;Walsh et al, 1976;Butler et al, 1977;Nemerya et al, 1996). In a recent study the mechanism of inactivation of human E1 was investigated using site-specific replacement of the three phosphorylation sites (Korotchkina and Patel, 2001a).…”

Section: Mechanism Of Inactivation Of Human E1 By Phosphorylation Of mentioning

confidence: 99%

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases

Patel

Korotchkina²

2001

Exp Mol Med

155

129

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This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.

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“…Many chemical modification studies of E1 have been performed. Cysteine [6–8], histidine [7], tryptophan [9, 10], arginine [11, 12]and lysine [13]residues have been suggested to be located in or near the active site, but the role of these residues in the mechanism of E1 is far from clear.…”

Section: Introductionmentioning

confidence: 99%

Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase

1998

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The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. The first component (E1) converts pyruvate to bound acetaldehyde using thiamine diphosphate (ThDP) and Mg 2+ as cofactors. There is no 3D structure of E1 available but those of other ThDP-dependent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an K K P L L P structure and the conserved Glu VW of the L L-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expressed in Escherichia coli and purified. Mutating Glu VW to glutamine, aspartate and alanine markedly reduces catalytic activity and the affinity for ThDP, consistent with a role as the catalytic glutamate.z 1998 Federation of European Biochemical Societies.

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Chemical modification of the essential arginine residues of pyruvate dehydrogenase prevents its phosphorylation by kinase

Cited by 5 publications

References 13 publications

The effect of phosphorylation on pyruvate dehydrogenase

The effect of phosphorylation on pyruvate dehydrogenase

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases

Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase

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