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by See-wing Chiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1980. / Bibliography: leaves 64-65.

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.

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Thesis (Ph.D.)--Case Western Reserve University, 2009 / Abstract Department of Mechanical and Aerospace Engineering Title from PDF (viewed on 14 April 2009) Available online via the OhioLINK ETD Center

Thesis (Ph. D.)--Johns Hopkins University, 1925. / Biography.

The kinetic study of the reactions, of the ethyl radical with the conjugated olefinic compounds has revealed the effect of various structual factors upon molecular reactivity. Acrylohitriie and its mono-methyl derivatives; cis- and trans-crotononitrile and methacrylonitrile were the substrates in this investigation. It was found that the addition of the ethyl radical to the C=C double bond was the most important reaction and that methathesis and addition to the CN triple bond could be neglected. Values for the energy of activation (E₇-½E₂) for cis- and trans-crotononitrile and methacrylonitrile were not distinguishable; 4.85 ±0.68, 4.69 ±0.96 and 4.61 ±0.67 respectively. The energy of activation for acrylonitrile for acrylonitrile was found to be 3.45 ±0.52. It appeared that the methyl group In cis- and trans-orotononitrile and in methacrylonitrile raised the energy of activation for addition from the value for acrylonitrile by about the same amount for each of the methyl derivatives. Simple correlations of the rate constants for the addition of the ethyl radical with the methyl affinities with various substrates, and with the monomer reactivity ratios for the copolymerization of various monomers with styrene were obtained. Copolymerizatlon reactions of cis- and trans-crotononitrile with styrene are also investigated and the polimerizability of these compounds is discussed. Some studies on the isomerization of cis- and trans- crotononitrile are also discussed. In this work, cis-croto-nonitrile was found to be thermally more stable than the trans isomer. / Science, Faculty of / Chemistry, Department of / Graduate

[No abstract available] / Science, Faculty of / Chemistry, Department of / Graduate