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.

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

1980 ◽  
Vol 187 (2) ◽  
pp. 393-401 ◽  
Author(s):  
Mary C. Ambrose-Griffin ◽  
Michael J. Danson ◽  
William G. Griffin ◽  
Geoffrey Hale ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Pyruvate Dehydrogenase Complex ◽  
Enzymic Activity ◽  
Complex Activity ◽  
Kinetics Of

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.

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 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 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 Lipoylation of the E2 components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli

1991 ◽  
Vol 277 (1) ◽  
pp. 153-158 ◽  
Author(s):  
L C Packman ◽  
B Green ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Catalytic Mechanism ◽  
Chemical Reduction ◽  
Pyruvate Dehydrogenase Complex ◽  
Exchange Chromatography ◽  
Lysine Residue ◽  
Multienzyme Complex ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The number of functional lipoyl groups in the dihydrolipoyl acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been re-assessed by means of a combination of protein-chemical and mass-spectrometric techniques. (1) After the complex had been treated with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, the lipoyl domains were excised from the complex, treated with NaBH4 and re-exposed to N-ethyl[2,3-14C]maleimide. All the chemically reactive lipoyl groups in the native complex were found to be catalytically active. (2) Proteolytic digests of the separated lipoyl domains were examined for the presence of the lipoylation-site peptide, GDKASME, with and without the lipoyl group in N6-linkage to the lysine residue. Only the lipoylated form of the peptide was detected, suggesting that all three lipoyl domains are fully substituted at this site. (3) The behaviour of each lipoyl domain was examined on ion-exchange chromatography in response to alkylation with 4-vinylpyridine after either chemical reduction of the lipoyl group with dithiothreitol or reductive acetylation by the pyruvate dehydrogenase complex in the presence of pyruvate. All three domains exhibited a quantitative shift in retention time, confirming that each domain was fully substituted by an enzymically reactive lipoyl group. (4) When subjected to electrospray mass spectrometry, each domain gave a mass consistent with a fully lipoylated domain, and no aberrant substitution of the target lysine residue was detected. The same result was obtained for the lipoyl domain from the E. coli 2-oxoglutarate dehydrogenase complex. (5) Previous widespread attempts to assess the number of functional lipoyl groups in the pyruvate dehydrogenase multienzyme complex, which have led to the view that a maximum of two lipoyl groups per E2 chain may be involved in the catalytic mechanism, are in error.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document