Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for essential histidine residue at the active site of lipoamide dehydrogenase

Biochemistry ◽  
1984 ◽  
Vol 23 (6) ◽  
pp. 1269-1274 ◽  
Author(s):  
S. Robert Adamson ◽  
James A. Robinson ◽  
Kenneth J. Stevenson
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Histidine Residue ◽  
Multienzyme Complex ◽  
Lipoamide Dehydrogenase

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.

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.

Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Biochemistry ◽  
1984 ◽  
Vol 23 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Peter N. Lowe ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Multienzyme Complex

Investigation of the mechanism of active site coupling in the pyruvate dehydrogenase multienzyme complex of Escherichia coli by protein engineering

1988 ◽  
Vol 202 (1) ◽  
pp. 97-106 ◽  
Author(s):  
John S. Miles ◽  
John R. Guest ◽  
Sheena E. Radford ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Protein Engineering ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Multienzyme Complex

scholarly journals Further Studies with Lipoamide Dehydrogenase Mutants of Escherichia coli k12

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 237-245 ◽  
Author(s):  
J. R. Guest ◽  
I. T. Creaghan
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Recombination Frequency ◽  
Regulatory Element ◽  
Multienzyme Complex ◽  
Wild Type ◽  
Mutant Site ◽  
Lipoamide Dehydrogenase ◽  
Distal Gene ◽  
P1 Transduction

The immunological properties of ten lipoamide dehydrogenase mutants of Escherichia coli were investigated with antiserum raised against purified lipoamide dehydrogenase. Seven mutants were CRM+ (cross-reacting material present) as they contained lipoamide dehydrogenase proteins exhibiting either complete or partial immunological identity with the wild-type protein. This indicates that at least seven of the mutations affect the lipoamide dehydrogenase structural gene (lpd). The remaining three mutants (CRM-) contained no detectable cross-reacting protein. None of the lpd mutations were sensitive to any of three different amber-suppressors. Genetic analysis by P1-transduction showed that all the lpd mutant sites were clustered very near the distal gene (aceF) of the ace region which specifies the dehydrogenase (aceE) and transacetylase (aceF) components of the pyruvate dehydrogenase multienzyme complex. Calculations based on the recombination frequency between an aceF mutant and the nearest lpd mutant site support the conclusion that apart from the possible presence of a regulatory element, the aceF and lpd genes are contiguous.

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