Interaction between the individual isoenzymes of pyruvate dehydrogenase kinase and the inner lipoyl-bearing domain of transacetylase component of pyruvate dehydrogenase complex

Abstract

Protein-protein interactions play an important role in the regulation of enzymic activity of pyruvate dehydrogenase kinase (PDK). It is generally believed that the binding of PDK to the inner lipoyl-bearing domain L2 of the transacetylase component E2 of pyruvate dehydrogenase complex largely determines the level of kinase activity. In the present study, we characterized the interaction between the individual isoenzymes of PDK (PDK1-PDK4) and monomeric L2 domain of human E2, as well as the effect of this interaction on kinase activity. It was found that PDK isoenzymes are markedly different with respect to their affinities for L2. PDK3 demonstrated a very tight binding, which persisted during isolation of PDK3-L2 complexes using size-exclusion chromatography. Binding of PDK1 and PDK2 was readily reversible with the apparent dissociation constant of approx. 10 microM for both isoenzymes. PDK4 had a greatly reduced capacity for L2 binding (relative order PDK3>PDK1=PDK2>PDK4). Monomeric L2 domain alone had very little effect on the activities of either PDK1 or PDK2. In contrast, L2 caused a 3-fold increase in PDK3 activity and approx. 37% increase in PDK4 activity. These results strongly suggest that the interactions between the individual isoenzymes of PDK and L2 domain are isoenzyme-specific and might be among the major factors that determine the level of kinase activity of particular isoenzyme towards the pyruvate dehydrogenase complex.

Regulation of Pyruvate Dehydrogenase Kinase 4 in the Heart through Degradation by the Lon Protease in Response to Mitochondrial Substrate Availability

“…Thus, dissociation of PDK4 from the PDH complex may expose a hydrophobic region and confer susceptibility to Lonmediated proteolysis. Properties that distinguish the short lived PDK4 from PDK1 and -2 that require investigation include lower binding affinity to the PDH complex and greater hydrophobic properties (17)(18)(19).…”

Recognition of the Inner Lipoyl-Bearing Domain of Dihydrolipoyl Transacetylase and of the Blood Glucose-Lowering Compound AZD7545 by Pyruvate Dehydrogenase Kinase 2

“…As discussed above, this could be due in part to potential folding problems, lower stability, or both. Usually, the addition of E2 to the phosphorylation cocktail is associated with a significant increase in PDHK2 activity (26,27). This phenomenon was rationalized in terms of (1) colocalization and mutual orientation of E1 and PDHK2 caused by their binding to E2, (2) E2-facilitated access of PDHK2 to the multiple copies of E1 attached to the E2 core, and (3) E2-dependent activation of PDHK2 (8).…”

“…Construction of the expression vectors for human E1, the human E2-E3BP subcomplex, Escherichia coli lipoyl-protein ligase A, human His 6 -L2 (amino acids Ser 127-Ile 214), human GST-L2 (amino acids Ser 127-Ile 214), and rat PDHK2 was described elsewhere (10,18,(25)(26)(27). Mutagenesis was conducted on previously described pPDHK2 and pGST-L2 vectors using appropriate oligonucleotide primers (18,27).…”

The C-terminal Hinge Region of Lipoic Acid-bearing Domain of E2b Is Essential for Domain Interaction with Branched-chain α-Keto Acid Dehydrogenase Kinase

“…The lipoyl-containing domains of the E2 cores function both as an anchor and a modulator for activities of the mitochondrial kinases. Studies with the lipoylated inner L2 domain have been complicated by the fact that the domain alone does not interact as efficiently as holo-E2p with the PDK isoforms, except for PDK3 (13,18). In this communication, we show that lip-LBD alone does not bind to BCK; however, the inclusion of various lengths of the previously neglected C-terminal hinge region results in the interaction between the lip-LBD constructs and BCK.…”

“…Constructs containing lip-LBD and various lengths of the C-terminal hinge region are able to bind to BCK to different degrees, as measured by a newly developed solubility-based assay and by isothermal titration calorimetry (ITC). The absence of relevant C-terminal hinge sequences may explain the previously observed weak binding of some PDK isoforms to the L2 domain of the PDC (13). More significantly, the inclusion of the C-terminal hinge region will facilitate investigations into the mechanism by which mitochondrial protein kinases, i.e.…”