Dissertation/Thesis Abstract

Kinetics and biological implications of three inositol phosphatase enzymes
by Wang, Yanling, Ph.D., Boston College, 2008, 247; 3310163
Abstract (Summary)

Inositol phosphates and the enzymes that dephosphorylate them play important and diverse roles in cells. In Archaeoglobus fulgidus, di-myo-inositol 1,1'-phosphate (DIP) is accumulated in cells grown above 80°C when they are subjected to salt and heat stress. An inositol monophosphatase (IMPase) plays a critical role in the biosynthesis of this solute by removing a phosphate on the phosphorylated DIP generated form L-I-1-P and CDP-inositol. Although other factors may also contribute to regulation of DIP synthesis in these cells, there was an 8-10-fold decrease in the Km of the IMPase for inositol phosphates between 75 and 85°C that correlates with the observed accumulation of DIP in cells. Between 55 and 75°C, Km values decreased 2.3-fold at most. 31P NMR studies confirmed that the affinity of inositol 1-phosphate for the enzyme was indeed weak (K D ≥ 5 mM) below but increased significantly at 80°C. In contrast, the IMPase from Methanococcus jannaschii, a hyperthermophilic organism in which DIP does not accumulate, had a low Km for I-1-P over the entire temperature range. A structural comparison of the two archaeal IMPases identified a hydrogen bonding network present in the active site of the A. fulgidus enzyme and not in the M. jannaschii IMPase, the disruption of which prevented the drop in Km at high temperatures.

The E. coli IMPase, the product of the suhB gene, is best known as a suppressor of temperature-sensitive growth phenotypes in E. coli. To gain insights into these biological diverse effects, we determined the structure of the SuhB R184A mutant protein. The SuhB dimer was organized much like other IMPases but with an altered interface suggesting that the presence of Arg-184 in the wild-type protein could shift the monomer-dimer equilibrium toward monomer. A variety of assays were used to show that SuhB binds RNA polymerase tightly and that the binding involves side chains in the dimer interface. The loss of sensitivity to RNA polymerase binding correlated strongly with loss of complementation of Δ suhB in vivo. Interactions of SuhB with nucleic acids were also examined. These interactions were not as strong as with RNA polymerase and appear to involve different parts of the protein. SuhB appears to prefer RNA that is cytosine-rich. However, the relevance of SuhB-nucleic acid interactions in vivo has yet to be established.

The last of the inositol phosphatases examined was PTEN, a lipid inositol phosphatase with a very important role in the PI3K/Akt signaling pathway that is critical for cell survival and upregulated in a variety of human cancer cell lines. PTEN opposes the action of phosphoinositide 3-kinase by dephosphorylating the signaling lipid phosphatidylinositol (3,4,5)-trisphosphate. A series of dioctanoylphosphatidylinositols were used to examine the kinetics of this enzyme and the structural requirements for inhibition by 3-deoxy-PI compounds. Specific activation by some isomers indicated the protein has an allosteric site for D-diC8PI and D-3-deoxy-diC 8PI(5)P binding. All the synthetic PI molecules were inhibitors of PTEN at high concentration (>0.5 mM), although the L-series were generally poorer inhibitors than the D-inositol lipids. The binding free energies calculated from computer simulations are consistent with the experimental IC50 values for all the inhibitors and provide strategies into constructing compounds that will specific enhance or inhibit PTEN.

Indexing (document details)
Advisor: Roberts, Mary F.
School: Boston College
School Location: United States -- Massachusetts
Source: DAI-B 69/04, Dissertation Abstracts International
Subjects: Biochemistry
Keywords: Di-myo-inositol phosphate, Inositol phosphatase, PTEN
Publication Number: 3310163
ISBN: 978-0-549-58591-6
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