Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and .alpha.-ketoglutarate dehydrogenase complexes of Escherichia coli

Stepp, Larry R.; Bleile, Dennis M.; McRorie, Donald K.; Pettit, Flora H.; Reed, Lester J.

doi:10.1021/bi00519a007

Cited by 64 publications

(54 citation statements)

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“…The finding that the lipoyl moieties in PDC and KGDC are located on a large; protruding segment (lipoyl domain) of the E2 subunit (9,29) led to the suggestion that mowment.of lipoyl domains and not simply rotation of lipoyllysyl moieties provides the means to span the physical gaps between catalytic sites on PDC and KGDC (10,11,14). On the basis oflimited proteolysis and proton NMR experiments, Packman etal.…”

Section: Discussionmentioning

confidence: 99%

“…Stepp et al (11) Insight into the extent and gedmetry of the relay network formed by lipoyl-lipoyl interactions on the succinyltransferase was obtained by observing the effect of different lipoyl-lipoyl interaction networks on the computed inactivation curves. Although caution must be exercised against too fine an interpretation of small variations in the computer simulations, one can conclude from Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Stepp et al (11) used trypsin and lipoamidase to examine the relationship between lipoic acid content and overall activity of PDC and KGDC. Limited proteolysis with trypsin releases the lipoyl domains from the E2 inner core, whereas lipoamidase releases only the lipoyl moieties, leaving the lipoyl domains otherwise intact.…”

mentioning

confidence: 99%

“…Due to the nature of interactions in the multienzyme complex, such data cannot be analyzed by standard kinetic treatments. The data were qualitatively interpreted to imply that in both complexes each E1 subunit is serviced by at least two lipoyl moieties that reside on two separate lipoyl domains and that in PDC a lipoicless lipoyl domain can act as a dead-end inhibitor (11).…”

mentioning

confidence: 99%

“…The reaction catalyzed by E1 is apparently rate-limiting in the overall reaction (16)(17)(18). The flexible lipoyl domains of the E2 subunits apparently permit the lipoyl moieties to rotate among the catalytic sites of E1, E2, and E3 (10,11,19) and also facilitate intramolecular transfer of electrons and acyl groups between lipoyl moieties (5,20). E3 is present in both PDC and KGDC Abbreviations: PDC, pyruvate dehydrogenase complex; KGDC, a-ketoglutarate dehydrogenase complex; E1, a-ketoglutarate dehydrogenase; E2, dihydrolipoamide succinyltransferase; E3, dihydrolipoamide dehydrogenase.…”

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confidence: 99%

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Evidence for a multiple random coupling mechanism in the alpha-ketoglutarate dehydrogenase multienzyme complex of Escherichia coli: a computer model analysis.

Hackert

Oliver

Reed

1983

Proc. Natl. Acad. Sci. U.S.A.

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A computer modeling system was used to analyze experimental data for inactivation of the Eacherichia coli a-ketoglutarate dehydrogenase complex accompanying release of lipoic acid residues by lipoamidase and by trypsin [Stepp, L. Each complex is composed of multiple copies of three enzymes: pyruvate dehydrogenase (EC 1.2.4.1) or a-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1), dihydrolipoamide acetyltransferase (EC 2.3.1.12) or dihydrolipoamide succinyltransferase (EC 2.3.1.61) (E2), and dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3). In both complexes the E2 component forms a structural core, composed of 24 subunits arranged with octahedral 432 symmetry in a cube-like particle, to which E1 and E3 are bound by noncovalent bonds (1, 2). Lipoic acid residues covalently attached to the e-amino group of lysine residues of E2 are thought to transfer intermediates between the catalytic sites of the component enzymes. There are two lipoyl moieties on each E2 subunit of the E. coli PDC, but only one lipoyl moiety per E2 subunit of the KGDC (3-8). Each E2 subunit consists of two principal domains: (i) a compact domain (subunit binding domain) that contains the subunit binding sites of E2, the binding sites for E, and E3, and the catalytic site for transacylation and (ii) a large, flexible extension that bears the covalently bound lipoyl moiety or moieties (lipoyl domain) (9, 10). The compact subunit binding domains of E2 form a closed structure, the E2 "inner" core, which determines the stoichiometry and quaternary organization of the E. coli PDC and KGDC.Stepp et al. (11) used trypsin and lipoamidase to examine the relationship between lipoic acid content and overall activity of PDC and KGDC. Limited proteolysis with trypsin releases the lipoyl domains from the E2 inner core, whereas lipoamidase releases only the lipoyl moieties, leaving the lipoyl domains otherwise intact. The results showed that release of lipoyl domains by trypsin and release of lipoyl moieties by lipoamidase proceeded at rates faster than the accompanying loss of overall activity of the two complexes. Of particular note was the finding that nearly one-half of the lipoyl domains in PDC could be removed before any appreciable loss of activity occurred. Due to the nature of interactions in the multienzyme complex, such data cannot be analyzed by standard kinetic treatments. The data were qualitatively interpreted to imply that in both complexes each E1 subunit is serviced by at least two lipoyl moieties that reside on two separate lipoyl domains and that in PDC a lipoicless lipoyl domain can act as a dead-end inhibitor (11).We have used computer modeling to quantitatively analyze the inactivation curves obtained for trypsin and lipoamidase treatments of PDC and KGDC in order to gain further insight into their mechanisms of action. Models that incorporate various patterns for lipoyl-E1, lipoyl-lipoyl, lipoyl-E2, and lipoyl-E3 interactions have been investigated. The results obtained with KGDC, the simpler of the two multienzyme complexes, a...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%