Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.

Abstract

Sequences located in the N‐terminal region of the high M(r) 2‐oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2‐oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N‐terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1′) and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This ‘lipoyl‐like’ domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1′ with trypsin both cause loss of overall OGDC function although the E1′ fragment retains full catalytic activity. Removal of this small N‐terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC.

Subunit Interactions in the Mammalian α-Ketoglutarate Dehydrogenase Complex

“…In addition, anti-E3BP (PDC) serum (B, lane 3) also recognizes E1 of OGDC (lane 4).Two minor degradation products of E3BP are also highlighted by the anti-E3BP serum (lane 3). As indicated, sequence similarities between E1 of OGDC and the E2 and E3BP subunits of PDC have been documented previously (13); this immunological evidence confirms that these polypeptides possess shared antigenic determinants and are likely to contain equivalent domains with common or overlapping functions.…”

“…Previous biochemical studies on mammalian OGDC detected significant sequence similarities between the N terminus of its E1 component and the E2 and E3BP subunits of eukaryotic PDCs corresponding to the N-terminal regions of their peripherally located lipoyl domains (13). Similar investigation of the N terminus of the E1Ј fragment indicated that these similarities extended into the interior of E1 and that the tryptic cleavage site was situated at a putative subunit-binding domain.…”

“…Complete disassembly of OGDC following this proteolytic event indicates that its subunit binding potential resides in this segment of its E1 component. A putative peripheral subunit-binding domain has been identified at the site of tryptic attack (13). It is now apparent that limited proteolysis also promotes release of E1, implying that sequences critical for its tight interaction with the E2 core FIG.…”

“…Earlier research has also suggested that this proteolytically sensitive site may lie within a putative subunit-binding domain (13), implying the potential for direct interaction between the E1 and E3 components in this complex. It has not been possible to test this hypothesis to date because of the tight association of E1 with the oligomeric E2 core as no methods were available for releasing the E1 enzyme from the E1/E2 subcomplex in active form.…”

“…Studies on mammalian OGDC in this laboratory, employing specific proteolysis and N-terminal sequence analysis, have identified a "lipoyl-like" region at the extreme N terminus of the E1 component with significant sequence similarity to the E3BP and E2 components of mammalian PDC (13). These similarities suggested that E1 of OGDC may perform some functions normally devolved to E2 in PDC and BCOADC.…”