scholarly journals Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1976 ◽  
Vol 159 (3) ◽  
pp. 677-682 ◽  
Author(s):  
M J Danson ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Acid Content ◽  
Polypeptide Chain ◽  
Multienzyme Complex ◽  
Covalently Bound

The reaction of two maleimides, N-ethylmaleimide and bis-(N-maleimidomethyl) ether, with the pyruvate dehydrogenase multienzyme complex of Escherichia coli in the presence of the substrate, pyruvate, was examined. In both cases, the reaction was demonstrated to be almost exclusively with the lipoate acetyltransferase component, and evidence is presented to show that the most likely sites of reaction are the lipoic acid residues covalently bound to this component. With both reagents the stoicheiometry of the reaction was measured: 2 mol of reagent reacted with each polypeptide chain of lipoate acetyltransferase, implying that each chain bears two functionally active lipolic acid residues. This observation can be reconciled with previous determinations of the lipoic acid content of the complex by allowing for the variability of the subunit polypeptide-chain ratio that can be demonstrated for this multimeric enzyme.

scholarly journals Polypeptide-chain stoicheiometry and lipoic acid content of the pyruvate dehydrogenase complex of Escherichia coli

1979 ◽  
Vol 177 (1) ◽  
pp. 129-136 ◽  
Author(s):  
G Hale ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Acid Content ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pyruvate Decarboxylase ◽  
Molar Ratios

The pyruvate dehydrogenase multienzyme complex was isolated from Escherichia coli grown in the presence of [35S]sulphate. The three component enzymes were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the molar ratios of the three polypeptide chains were determined by measurement of the radioactivity in each band. The chain ratio of lipoamide dehydrogenase to lipoate acetyltransferase approached unity, but there was a molar excess of chains of the pyruvate decarboxylase component. The 35S-labelled complex was also used in a new determination of the total lipoic acid content. It was found that each polypeptide chain of the lipoate acetyltransferase component appears to bear at least three lipoyl groups.

scholarly journals Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 733-740 ◽  
Author(s):  
R N Perham ◽  
G C K Roberts
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Proton Nuclear Magnetic Resonance ◽  
Resonance Spectroscopy ◽  
Multienzyme Complex ◽  
Oxoglutarate Dehydrogenase ◽  
Polypeptide Chains ◽  
Dehydrogenase Complex

The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.

scholarly journals The role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 505-511 ◽  
Author(s):  
M J Danson ◽  
G Hale ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Chemical Modification ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Enzymic Activity ◽  
Multienzyme Complex ◽  
Dihydrolipoic Acid ◽  
Lipoamide Dehydrogenase

Two lipoic acid residues on each dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were found to undergo oxidoreduction reactions with NAD+ catalysed by the lipoamide dehydrogenase component. It was observed that: (a) 2 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of acetyl-SCoA and NADH; (b) 4 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of NADH; (c) between 1 and 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with acetyl-SCoA plus NADH; (d) 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with pyruvate either before or after many catalytic turnovers through the overall reaction. There was no evidence to support the view that only half of the dihydrolipoic acid residues can be reoxidized by NAD+. However, chemical modification of lipoic acid residues with N-ethylmaleimide was shown to proceed faster than the accompanying loss of enzymic activity under all conditions tested, which indicates that not all the lipoyl groups are essential for activity. The most likely explanation for this result is an enzymic mechanism in which one lipoic acid residue can take over the function of another.

scholarly journals Segmental structure and protein domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Genetic reconstruction in vitro and 1H-n.m.r. spectroscopy

1987 ◽  
Vol 247 (3) ◽  
pp. 641-649 ◽  
Author(s):  
S E Radford ◽  
E D Laue ◽  
R N Perham ◽  
J S Miles ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Enzyme Complex ◽  
Multienzyme Complex ◽  
Wild Type ◽  
Dihydrolipoamide Dehydrogenase ◽  
Core Domain

A deletion in vitro can be made in the aceEF-lpd operon encoding the pyruvate dehydrogenase multienzyme complex of Escherichia coli, which causes deletion of two of the three homologous lipoyl domains that comprise the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain. An active complex is still formed and 1H-n.m.r. spectroscopy of this modified complex revealed that many of the unusually sharp resonances previously attributed to conformationally mobile segments in the wild-type E2p polypeptide chains had correspondingly disappeared. A further deletion was engineered in the long (alanine + proline)-rich segment of polypeptide chain that linked the one remaining lipoyl domain to the C-terminal half of the E2p chain. 1H-n.m.r. spectroscopy of the resulting enzyme complex, which was also active, revealed a further corresponding loss in the unusually sharp resonances observed in the spectrum. These experiments strongly support the view that the sharp resonances derive, principally at least, from the three long (alanine + proline)-rich sequences which separate the three lipoyl domains and link them to the C-terminal half of the E2p chain. Closer examination of the 400 MHz 1H-n.m.r. spectra of the wild-type and restructured complexes, and of the products of limited proteolysis, revealed another sharp but smaller resonance. This was tentatively attributed to another, but smaller, (alanine + proline)-rich sequence that separates the dihydrolipoamide dehydrogenase-binding domain from the inner core domain in the C-terminal half of the E2p chain. If this sequence is also conformationally flexible, it may explain previous fluorescence data which suggest that dihydrolipoamide dehydrogenase bound to the enzyme complex is quite mobile. The acetyltransferase active site in the E2p chain was shown to reside in the inner core domain, between residues 370 and 629.

scholarly journals Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1978 ◽  
Vol 175 (1) ◽  
pp. 193-198 ◽  
Author(s):  
M J D Danson ◽  
E A Hooper ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Pyruvate Decarboxylase ◽  
Enzyme Complex ◽  
Multienzyme Complex ◽  
Thiol Groups ◽  
Specific Reaction ◽  
Good Agreement

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.

scholarly journals Amino acid sequence around lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1980 ◽  
Vol 187 (3) ◽  
pp. 905-908 ◽  
Author(s):  
G Hale ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Multienzyme Complex ◽  
Dehydrogenase Complex

Amino-acid sequences around two lipoic acid residues in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli were investigated. A single amino acid sequence of 13 residues was found. A repeated amino acid sequence in the lipoate acetyltransferase chain might explain this result.

scholarly journals Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1976 ◽  
Vol 155 (2) ◽  
pp. 429-432 ◽  
Author(s):  
M C. Ambrose ◽  
R N. Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Resonance Spectrum ◽  
Protein Molecule ◽  
Multienzyme Complex ◽  
Label Study ◽  
Spin Resonance

The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl. The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule. This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism.

Acetylatable lipoic acid residues interact directly with lipoamide dehydrogenase in the pyruvate dehyrogenase multienzyme complex of Escherichia coli

1986 ◽  
Vol 64 (3) ◽  
pp. 250-255 ◽  
Author(s):  
S. Robert Adamson ◽  
Charles F. B. Holmes ◽  
Kenneth J. Stevenson
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Functional Role ◽  
Reduced Form ◽  
Multienzyme Complex ◽  
Chemically Modified ◽  
The Third ◽  
Lipoamide Dehydrogenase

The proposal that the lipoate acetyltransferase component (E2) of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli contains three covalently bound lipoyl residues, one of which acts to pass reducing equivalents to lipoamide dehydrogenase (E3), has been tested. The PD complex was incubated with pyruvate and N-ethylmaleimide, to yield an inactive PD complex containing lipoyl groups on E2 with the S6 acetylated and the S8H irreversibly alkylated with N-ethylmaleimide. This chemically modified form would be expected to exist only on two of the three proposed lipoyl groups. The third nonacetylatable lipoyl group, which is proposed to interact with E3, would remain in its oxidized form. Reaction of the N-ethylmaleimide-modified PD complex with excess NADH should generate the reduced form of the proposed third nonacetylatable lipoyl group and thereby make it susceptable to cyclic dithioarsinite formation with bifunctional arsenicals (BrCH2CONHPhAsCl2; BrCH2[14C]CONHPhAsO). Once "anchored" to the reduced third lipoyl group via the —AsO moiety, these reagents would be delivered into the active site of E3 by the normal catalytic process of the PD complex where the BrCH2CONH— group inactivates E3. Whereas the E3 component of native PD complex is inactivated by the bifunctional reagents in the presence of excess NADH (owing to the above delivery process), the E3 component of the PD complex modified with N-ethylmaleimide in the presence of pyruvate is not inhibited. The results indicate that acetylatable lipoyl residues interact directly with E3 and do not support a functional role for a proposed third lipoyl residue.

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