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

Abstract

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...

Chromosomal Amplification of the Escherichia coli lipB Region Confers High-Level Resistance to Selenolipoic Acid

“…Therefore, when the enzymatic and in vivo data are considered in context, they indicate that attachment of lipoic acid in place of Se-lip to a few percent of the 2-oxo acid dehydrogenase complexes would be sufficient for growth. This picture is not only consistent with the behavior of the slr7 mutant but argues strongly that the nonlinear mechanism proposed for the 2-oxo acid dehydrogenases (1,5,6,10,15,30,36) operates in vivo. In order to further test this argument, it would be advantageous to measure the extent of lipoic acid attachment in the slr7 mutant grown with Se-lip.…”

“…The structures and enzyme mechanism of the 2-oxo acid dehydrogenases are known to combine to give a highly nonlinear relationship between the degree of protein lipoylation and the in vitro activity of the 2-oxoacid dehydrogenase complex (1,5,6,10,15,30,36). The rate-limiting step in the reaction mechanism is decarboxylation of the 2-oxo acid (Fig.…”

“…Moreover, a single lipoyl domain is able to productively access many active sites within these very large protein complexes. That is, the lipoyl domain of one E2 subunit can be acetylated by E1 subunits bound to different E2 subunits; therefore, there is only a weak dependence of the overall activity of the complex on the level of protein-bound lipoic acid (1,5,6,10,15,30,36). These conclusions result from a variety of experimental approaches in several laboratories that removed or inactivated major fractions of the lipoyl domains of a 2-oxo acid dehydrogenase complex.…”

The α‐Ketoglutarate Dehydrogenase Complex

“…28 The structure and mechanism of action of the bacterial E2k core has been worked out in detail. 29–31 E1k and E3 units adhere, noncovalently, to the E2k core. In mammalian KGDHC, some studies suggest that the E1k protein appears to bind more tightly to the E2k core than does the E3 protein.…”

“…This linkage provides a flexible arm about 140 μm long, which can rotate among the E1k, E2k, and E3 subunits, as a “swinging arm.” A “multiple random coupling mechanism” has been proposed, based on computer modeling of KGDHC from E. coli . 29…”