S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) peptide regions of its catalytic kinase domain (residues 1-398). In addition to its kinase domain, S6K1 contains a C-terminal autoinhibitory domain (AID; residues 399-502), which prevents T-loop and HM phosphorylation and autoinhibition is relieved on multi-site Ser-Thr phosphorylation of the AID (S411, S418, T421, and S424). The fully activated catalytic kinase domain construct, His6-S6K1 alphaII(∆AID)-T389E (activity = 250 nmol/min/mg) was generated by baculovirus-mediated expression and purification from Sf9 insect cells that were coinfected with recombinant baculovirus expressing the catalytic kinase domain of PDK1 [His6-PDK1(∆PH)]. The kinetic mechanism of fully active His6-S6K1 alphaII(∆AID)-T389E for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA) was determined. Two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, while pre-steady state kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the enzyme's affinity for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme. We propose that ordered binding of ATP causes partial unfolding of enzyme residues, which unmask the peptide substrate binding epitope. Next, the kinetic mechanism of PDK1 for catalyzing T229 phosphorylation of S6K1 (native and T389E mutant forms) was determined. Surprisingly, we found that His6-PDK1(∆PH) effectively and specifically phosphorylates T229 of His6-S6K1 alphaII(∆AID), regardless of whether a negative charge is localized at residue 389. Steady-state kinetic studies revealed S6K1 alphaII to be a competitive inhibitor of ATP, thereby enforcing an Ordered Bi Bi mechanism whereby ATP must bind first. Kinetic studies further revealed exceptionally slow bimolecular association of S6K1 alphaII substrate to form the productive ternary complex that catalyzes S6K1 alphaII T229 phosphorylation, indicating a high degree of nonproductive binding events. In this regard, the T389E mutant exhibited a two-fold increased efficiency of productive binding over native S6K1 alphaII. Finally, to investigate the regulatory role of C-terminal AID of S6K1, we developed and optimized protocols for efficient AID expression and purification. Consistent with computer predictions, aberrant mobilities in both SDS-PAGE and size-exclusion chromatography, as well as low chemical shift dispersion in 1H-15N HSQC NMR spectra, indicated purified recombinant AID to be largely unfolded. Yet, trans-addition of purified AID effectively inhibited PDK1-catalyzed T-loop phosphorylation of a catalytic kinase domain construct of S6K1. Using an identical purification protocol, similar protein yields of a tetraphospho-mimic mutant AID(D2ED) construct were obtained; and this construct displayed only weak inhibition of PDK1-catalyzed T229 phosphorylation.

Würzburg, Univ., Diss., 2000. / Erscheinungsjahr an der Haupttitelstelle: 2000. Computerdatei im Fernzugriff.

Hannover, Universiẗat, Diss., 2004.

Würzburg, Universiẗat, Diss., 2000. / Erscheinungsjahr an der Haupttitelstelle: 2000.

Hannover, Universiẗat, Diss., 2004.

Note:

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Human pyruvate kinase performs the last step in glucose glycolysis in all cells and organisms and can be a key regulator of glycolytic flux. Pyruvate produced by PYK is transported into the mitochondria to fuel the TCA cycle, which enables the production of ATP; the main energy source of the cell. Human PYK contains four isoforms: M1 (found in muscle, heart and brain), M2 (in foetal cells and tumours), L (liver), and R (red blood cells) PYK. M2PYK plays a crucial role in tumour cell proliferation; by down-regulating metabolic flux, upstream metabolites can be used for protein and DNA synthesis. Reprogramming the metabolism of fast proliferating cells is called the 'Warburg effect'. The biological relevance of the different isoform activities is also discussed. For example RPYK in red blood cells is exposed to slowly altering metabolite concentrations, especially after intestinal absorption in plasma and RBCs uptake some of the metabolites. This thesis describes biochemical and biophysical studies of human M1PYK, M2PYK, LPYK, and RPYK. PYK is allosterically regulated by a range of metabolites. A comparative enzyme kinetics study of the four isoforms was performed to examine the mechanisms of activation and inhibition of these small molecule regulators, including all 20 amino acids and the thyroid hormone T3. The redox state of the environment was also found to be an important regulator of PYK activity. All four PYK isoforms were successfully expressed and purified. Interestingly, only M2PYK and RPYK were strongly regulated by amino acids and metabolites. We also found that the redox state regulates the activity of all four PYK isoforms as well as the sensitivity of M2PYK in response to natural regulators. These studies also confirmed the dissociation of tetrameric PYK into inactive monomers as an important mechanism of regulation, particularly for M2PYK activity. Nuclear magnetic resonance (NMR) and Small-angle X-ray scattering (SAXS) studies were performed to investigate the conformational behaviour of PYK isoforms in solution and to compare the effects of ligand binding. NMR data of all four isoforms reveal a conserved binding mechanism between isoforms and specific amino acids. SAXS data of all four isoforms demonstrate that ligands affect tetramerisation of PYK isoforms.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: MAPK cascades in mammals & yeast. Includes bibliographical references (p. 122-141). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (p. 150-177). Also available online through Digital Dissertations.