Enzymatic properties of Sir2 proteins.
Published: 2006
Language: eng
Number of pages: 232
ISBN-13: 9780494217733
Description: Although many structural and enzymatic studies of Sir2 proteins have been reported, how substrate recognition is achieved by this family of enzymes is not known. Here I use in vitro assays and a variety of potential substrates to examine the substrate specificity of Hst2. I show that Hst2 is specific for acetyl-lysine within proteins; it does not deacetylate small polycations such as acetyl-spermine or acetylated amino-termini of proteins. Furthermore, Hst2 displays conformational rather than sequence specificity, preferentially deacetylating acetyl-lysine within unstructured regions of proteins. Results suggest that this lack of conformation may be a general requirement for substrate recognition in the Sir2 family.The Sir2 family of NAD-dependent deacetylases is highly conserved and functions in silencing, control of lifespan, apoptosis, and many other cellular processes. Since the discovery of their NAD-dependent deacetylase activity, researchers have aimed at uncovering the mode of substrate binding and catalysis of these enzymes. The studies presented herein focus on uncovering the biochemical mechanisms underlying Sir2 enzymatic activity. To this end, I performed  in vitro studies of the yeast homolog Hst2. General biophysical and biochemical characterization of Hst2, including structural and kinetic analyses were done. These studies indicate that Hst2-mediated deacetylation proceeds via an ordered sequential bisubstrate mechanism in which the acetylated substrate binds first, followed by the coenzyme beta-NAD+. The reaction generates a unique product, O-acetyl-ADP-ribose.Structural and biochemical studies have led to several proposed reaction mechanisms for Sir2 enzymes, yet the exact catalytic steps remain unclear. Using acetyl-lysine substrate analogs I demonstrate that the Hst2 reaction proceeds via an initial SN2-type mechanism with the direct formation of an ADP-ribose-acetyl-lysine intermediate. Kinetic studies further suggest that ADP-ribose inhibits the Hst2 reaction in a biologically relevant manner. Furthermore, biochemical and kinetic analyses of point mutants clarify the role of several conserved core domain residues in substrate binding and catalysis. These findings bring us one step closer to understanding Sir2 activity and may provide a useful platform for the design of Sir2-specific inhibitors for analysis of Sit-2 function and possibly therapeutic applications.
Notes: Source: Dissertation Abstracts International, Volume: 68-01, Section: B, page: 0273.

Thesis (Ph.D.)--University of Toronto, 2006.

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