Antibodies against an inter‐domain segment of polypeptide chain inhibit active‐site coupling in the pyruvate dehydrogenase multienzyme complex

Abstract

A synthetic peptide, AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA, proved to be an efficient and specific immunogen in rabbits. The amino acid sequence of the peptide is identical to that of the inter-domain region (PEP3) linking the innermost of the three lipoyl domains to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coll. Fab fragments from anti-PEP3 antibodies selectively inhibited active-site coupling in the complex without affecting the individual activities of the three component enzymes, highlighting the role of the inter-domain regions as flexible linkers in catalysis.

Isolation and characterization of lipoylated and unlipoylated domains of the E2p subunit of the pyruvate dehydrogenase complex of Escherichia coli

“…This shows that the antisera are specifically interfering with the acetyl-carrying function of the corresponding lipoyl domain. Studies with sera raised against an interdomain linker have given similar results (Radford et al, 1989). In the sedimented samples it was apparent that the antisera removed the PDH complex, Elp and E2p activities, and part of the E3 activity, before affecting the ODH complex and its associated E3 activity (Fig.…”

Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein

“…Such a mechanism clearly rests on the interdigitation of the lipoyl domains between the peripheral El and E3 subunits and the conformational flexibility in the interdomain segments of the E2 polypeptide chains that permits these domains to link the different and widely separated active sites. This has been emphasized by the demonstration that Fab fragments of antibodies raised against the interdomain segment that joins the innermost lipoyl domain to the E3-binding domain in the E. coli E2p chain inhibit the overall PDH complex activity (and the formation of the acetyl-enzyme intermediate) without effect on the individual part-reactions catalyzed by the El, E2, or E3 components (Radford et al, 1989b). This is most simply explained by the Fab fragments acting, like wedges, to prevent the movement of the lipoyl domains necessary for active-site coupling.…”

Protein–protein interactions in the pyruvate dehydrogenase multienzyme complex: dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase

“…It forms the inner core of the multienzyme complex and is also responsible for binding E1p in the octahedral complexes. These three domains are connected to each other by long and highly flexible linker regions, thereby allowing the lipoyl moiety to 'visit' all three active sites during catalysis [14][15][16][17][18][19].…”