Year: 2009
Type: dissertation
Abstract: The primary role of pyruvate dehydrogenase kinase (PDK) is to regulate the activity of \npyruvate dehydrogenase complex (PDC) with respect to the metabolic clearance of \nglucose via a phosphorylation mechanism. Therefore, inhibition of PDK is predicted to \nbe important in the treatment of diabetes. ADP binds to the active site of PDK; and has \nbeen shown to inhibit PDK activity. This research work was aimed at studying one of the \nisoforms of PDK, specifically the rodent form, PDK4 (rPDK4) and further elucidating the \nbinding properties of ADP to rPDK4. \nThe hypothetical structure of rPDK4 was modelled based on the coordinates of the \npublished rPDK2 structure. The overall structural topology of rPDK2 appears to be \npreserved in rPDK4. Further, the ADP binding site between rPDK2 and rPDK4 was \nconserved at both the primary and tertiary level, and this suggested the mechanism of ADP binding to rPDK2 would be similar to that of rPDK4. \nA histidine tagged-rPDK4 protein was expressed and purified using affinity and gel \nfiltration chromatography. It was determined to be a homodimer (97 kDa) comprising \ntwo identical subunits. The rPDK4 protein was further identified to be rPDK4 using \nWestern blot analysis as it reacted positively with an anti-rPDK4 monoclonal antibody. \nThe purified rPDK4 protein contained kinase activity since it was able to undergo \nautophosphorylation and subsequently phosphorylate a peptide that contained the E1a \nsubunit sites that are known to be phosphorylated by PDKs. The structure of rPDK4 \nprotein was also characterised using circular dichroism and fluorescence spectroscopy.The spectroscopic data of rPDK4 was consistent with published data on the structure of \nrPDK4. \nThe binding of ADP and ATP was studied using fluorescence quenching as both these \nligands quench the intrinsic tryptophan fluorescence of rPDK4. The dissociation constant \n(Kd) values for ADP and ATP were determined to be 37 and 17.4 μM, respectively. The \nmoderate affinity binding of ATP will greatly favour the exchange of ADP for a molecule \nof ATP to prevent PDK inhibition by ADP.
Author: Thandeka Khoza
Language: English
Subfield: Biochemistry
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2158083811