Use of substrate analogs and mutagenesis to study substrate binding and catalysis in the Sir2 family of NAD-dependent protein deacetylases

The Journal of Biological Chemistry
Ahlia N Khan, Peter N Lewis

Abstract

The Sir2 family of enzymes is highly conserved throughout evolution and functions in silencing, control of life span, apoptosis, and many other cellular processes. Since the discovery of the NAD-dependent deacetylase activity of Sir2 proteins, there has been a flurry of activity aiming to uncover the mode of substrate binding and catalysis. Structural and biochemical studies have led to several proposed reaction mechanisms, yet the exact catalytic steps remain unclear. Here we present in vitro studies of yeast homolog Hst2 that shed light on the mechanism of Sir2 proteins. Using acetyl-lysine substrate analogs, we 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. Through biochemical and kinetic analyses of point mutants, we also clarify the role of several conserved core domain residues in substrate binding, stabilization of the ADP-ribose-acetyl-lysine intermediate, and catalysis. These findings bring us a few steps closer to understanding Sir2 activity and may provide a useful platform for the design of Sir2-specific inh...Continue Reading

References

Feb 15, 1988·European Journal of Biochemistry·P N LewisS Chan
Mar 1, 2000·Trends in Biochemical Sciences·C CombetG Deléage
Jan 3, 2001·Proceedings of the National Academy of Sciences of the United States of America·J C Tanny, D Moazed
Jun 28, 2001·Nature Structural Biology·M S FinninN P Pavletich
Oct 24, 2001·Cell·H VaziriR A Weinberg
Dec 26, 2001·Proceedings of the National Academy of Sciences of the United States of America·A BedalovJ A Simon
Jul 2, 2002·The Journal of Biological Chemistry·Jeong-Ho ChangYunje Cho
Nov 1, 2002·Molecular Cell·Jose L AvalosCynthia Wolberger
Mar 7, 2003·Molecular Cell·Brian J NorthEric Verdin
Apr 3, 2003·Nature Reviews. Molecular Cell Biology·Siavash K Kurdistani, Michael Grunstein
Dec 1, 1964·Analytical Biochemistry·R F ITZHAKI, D M GILL
Sep 23, 2003·Nature Structural Biology·Kehao ZhaoRonen Marmorstein
Jan 20, 2004·Molecular and Cellular Biology·Christopher M GalloJeffrey S Smith
Mar 17, 2004·Molecular Cell·José L AvalosCynthia Wolberger
Jun 11, 2004·Annual Review of Biochemistry·Gil Blander, Leonard Guarente
Sep 9, 2004·The Biochemical Journal·Mathias Ziegler, Marc Niere
Jan 11, 2005·The Journal of Biological Chemistry·Gil BlanderLeonard Guarente
Mar 4, 2005·The International Journal of Biochemistry & Cell Biology·Mirella L Meyer-FiccaMyron K Jacobson
Jul 30, 2005·Science·Dudley W LammingDavid A Sinclair
Sep 1, 2005·The Journal of Biological Chemistry·Ahlia N Khan, Peter N Lewis

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Jul 8, 2008·Biochimica Et Biophysica Acta·Brian C Smith, John M Denu
Jan 29, 2008·Molecular and Biochemical Parasitology·Subhra Prakash ChakrabartyHemalatha Balaram
Jun 16, 2015·The Journal of Organic Chemistry·Joseph M GriffinMichael I Page
Oct 23, 2008·Chemistry & Biology·Brian C SmithJohn M Denu
Feb 12, 2015·Langmuir : the ACS Journal of Surfaces and Colloids·Indah Nurita KurniasihAndreas Mohr
Jul 6, 2014·Biochemical and Biophysical Research Communications·You Hee ChoiKwang Youl Lee
May 3, 2011·The Journal of Organic Chemistry·Karl VoigtritterBruce H Lipshutz
Oct 16, 2009·Journal of the American Chemical Society·Arkady KrasovskiyBruce H Lipshutz
Dec 11, 2013·Journal of Chemical Education·Bruce H LipshutzAnna G Wenzel
May 10, 2011·The Journal of Organic Chemistry·Bruce H LipshutzRobert C Gadwood
May 9, 2015·Chemical Society Reviews·Bing ChenWeiping Zheng
May 16, 2009·Expert Opinion on Therapeutic Patents·Francisco J Alcaín, José M Villalba
Sep 16, 2010·Molecular BioSystems·Brett M Hirsch, Weiping Zheng
Oct 24, 2007·The Journal of Biological Chemistry·Brian C Smith, John M Denu
Apr 16, 2013·The Journal of Physical Chemistry Letters·Yawei ShiYingkai Zhang
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