LEADER 03465ctm a22003017 4500
008 030226s2002 xx a bm 000 0 eng d
z| 9780493929101 q| (electronic)
a| PU c| PU
a| Kashlan, Ossama Bassam.
a| A comprehensive model for the allosteric regulation of mammalian ribonucleotide reductase / c| Ossama Bassam Kashlan.
a| xx, 298 p. : b| ill., (some col.) ; c| 29 cm.
a| Supervisor: Barry S. Cooperman.
a| Thesis (Ph.D. in Chemistry) -- University of Pennsylvania, 2002.
a| Includes bibliographical references.
a| University Microfilms order no.: 3073019.
a| Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present a comprehensive and quantitative model for allosteric control of mRR enzymatic activity based on molecular mass, ligand binding and enzyme activity studies. In this model, nucleotide binding to the specificity site (s-site) drives formation of R12, ATP or dATP binding to the adenine-specific site (a-site) results in formation of tetramer R14a which isomerizes to the more stable R14b , and ATP binding to the newly described hexamerization site (h-site) drives formation of R16. The R22 complexes of R1 2, R14a, and R16 are enzymatically active, whereas the R22 comples of R14b is not. Thus, a key aspect of the down regulation of RR enzymatic activity is the ability of dATP or ATP binding to the a-site to drive not only R1 tetramer formation, but also the conversion of R14a to R14b, and it is ATP binding to the h-site which accounts for its activating properties at high (>1 mM) concentration. The D57N variant of mR1 is not inhibited by dATP because of a block in the formation of R14b, but not of R14a or R16. The new model revises and improves upon an earlier phenomenological model (Thelander and Reichard, 1979) (the 'RT' model) which ignores aggregation state changes and the h-site, and incorrectly rationalizes ATP activation vs. dATP inhibition as reflecting different functional consequences of ATP vs. dATP binding to the a-site. Our results suggest that the R1 6R26 heterohexamer is the major active form of the enzyme in mammalian cell cytoplasm, where [ATP] is the primary modulator of enzyme activity, coupling the rate of DNA biosynthesis with the energetic state of the cell. However, it remains possible that the heterodimer formed by complexation of R12 with p53R2 is the active form of the enzyme in the nucleus. Using the crystal structure of the Escherichia coli R1 hexamer as a model for the mR1 hexamer, a scheme is presented that rationalizes the slow isomerization of the tetramer form and suggests an explanation for the low enzymatic activity of tetramers complexed with R2. The similar specific activities of R12R22 and R16R26 are inconsistent with a proposed model for R22 docking with R1 2 (Uhlin and Eklund, 1994) and an alternative model is proposed.
a| Penn dissertations x| Chemistry.
a| Chemistry x| Penn dissertations.
a| Cooperman, Barry S., e| advisor.
a| University of Pennsylvania.