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

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 Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

2008 ◽  
Vol 105 (4) ◽  
pp. 1158-1163 ◽  
Author(s):  
S. Kale ◽  
G. Ulas ◽  
J. Song ◽  
G. W. Brudvig ◽  
W. Furey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Multienzyme Complex ◽  
Efficient Coupling

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.

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