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ALLOSTERIC REGULATION OF THE CANCER ASSOCIATED MITOCHONDRIAL GLUTAMINASE GAC

Author
Li, Yunxing
Abstract
Glutamine-derived carbon becomes available for anabolic biosynthesis in cancer cells via the hydrolysis of glutamine to glutamate, as catalyzed by GAC, a splice variant of kidney-type glutaminase (GLS). Thus, there is significant interest in understanding the regulation of GAC activity, with the suggestion being that higher order oligomerization is required for its activation. We used x-ray crystallography, together with site directed mutagenesis, to determine the minimal enzymatic unit capable of robust catalytic activity. Mutagenesis of the helical interface between the two pairs of dimers comprising a GAC tetramer yielded a non-active, GAC dimer whose x-ray structure displays a stationary loop (‘activation loop’) essential for coupling the binding of allosteric activators like inorganic phosphate to catalytic activity. Further mutagenesis that removed constraints on the activation loop yielded a constitutively active dimer, providing clues regarding how the activation loop communicates with the active site, as well as with a peptide segment that serves as a ‘lid’ to close off the active site following substrate binding. Our studies show that the formation of large GAC oligomers is not a pre-requisite for full enzymatic activity. They also offer a mechanism by which the binding of activators like inorganic phosphate enables the activation loop to communicate with the active site to ensure maximal rates of catalysis, and promotes the opening of the lid to achieve optimal product release. Moreover, these findings provide new insights into how other regulatory events might induce GAC activation within cancer cells.
Date Issued
2018-05-30Subject
Cancer metabolism; Biophysics; Glutaminase; mitochondrion
Committee Chair
Cerione, Richard A.
Committee Member
Baird, Barbara Ann; Sondermann, Holger
Degree Discipline
Biophysics
Degree Name
Ph. D., Biophysics
Degree Level
Doctor of Philosophy
Rights
Attribution 4.0 International
Rights URI
Type
dissertation or thesis
Except where otherwise noted, this item's license is described as Attribution 4.0 International