Subunit Interactions in the Mammalian α-Ketoglutarate Dehydrogenase Complex: EVIDENCE FOR DIRECT ASSOCIATION OF THE α-KETOGLUTARATE DEHYDROGENASE AND DIHYDROLIPOAMIDE DEHYDROGENASE COMPONENTS

R Graham Mccartney,Jacqueline E Rice,Sanya J Sanderson,Victoria Bunik,Heather Lindsay,J Gordon Lindsay

doi:10.1074/jbc.273.37.24158

R Graham Mccartney, Jacqueline E Rice + Show 4 more

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https://doi.org/10.1074/jbc.273.37.24158

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Abstract

Selective tryptic proteolysis of the mammalian alpha-ketoglutarate dehydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its alpha-ketoglutarate dehydrogenase (E1) component, which promotes the dissociation of the dihydrolipoamide dehydrogenase (E3) enzyme and also a fully active E1' fragment. Similarities between the N-terminal region of E1 and the dihydrolipoamide acetyltransferase (E2) and E3-binding components (E3BP) of the pyruvate dehydrogenase complex are highlighted by the specific cross-reactivities of subunit-specific antisera. Analysis of the pattern of release of E1 and E1' polypeptides from the OGDC during tryptic inactivation suggests that both polypeptide chains of individual E1 homodimers must be cleaved to permit the dissociation of the E1 and E3 components. A new protocol has been devised that promotes E1 dissociation from the oligomeric dihydrolipoamide succinyltransferase (E2) core in an active state. Significant levels of overall OGDC reconstitution could also be achieved by re-mixing the constituent enzymes in stoichiometric amounts. Moreover, a high affinity interaction has been demonstrated between the homodimeric E1 and E3 components, which form a stable subcomplex comprising single copies of these two enzymes.

Highlights

Selective tryptic proteolysis of the mammalian ␣-ketoglutarate dehydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its ␣-ketoglutarate dehydrogenase (E1) component, which promotes the dissociation of the dihydrolipoamide dehydrogenase (E3) enzyme and a fully active E1؅ fragment
Sequence similarities between E1 of OGDC and the E2 and E3-binding components (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
Selective Proteolysis of OGDC—Previous limited proteolysis and N-terminal sequencing studies have identified a lipoyl-like domain at the N terminus of the E1 component of mammalian OGDC

Summary

Introduction

Selective tryptic proteolysis of the mammalian ␣-ketoglutarate dehydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its ␣-ketoglutarate dehydrogenase (E1) component, which promotes the dissociation of the dihydrolipoamide dehydrogenase (E3) enzyme and a fully active E1؅ fragment. In higher eukaryotes the identification of an additional component of PDC, E3-binding protein, (E3BP, formerly protein X) with similarities in domain structure to E2 suggested a possible devolution of subunit function [6, 7].

Results

Conclusion