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J. Biol. Chem., Vol. 282, Issue 51, 37256-37265, December 21, 2007

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Acetyl-lysine Analog Peptides as Mechanistic Probes of Protein Deacetylases^*

Brian C. Smith and John M. Denu¹

From the Departments of Chemistry and Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Class III histone deacetylases (Sir2 or sirtuins) catalyze the NAD⁺-dependent conversion of acetyl-lysine residues to nicotinamide, 2'-O-acetyl-ADP-ribose (OAADPr), and deacetylated lysine. Class I and II HDACs utilize a different deacetylation mechanism, utilizing an active site zinc to direct hydrolysis of acetyl-lysine residues to lysine and acetate. Here, using ten acetyl-lysine analog peptides, we have probed the substrate binding pockets of sirtuins and investigated the catalytic differences among sirtuins and class I and II deacetylases. For the sirtuin Hst2, acetyl-lysine analog peptide binding correlated with the hydrophobic substituent parameter with a slope of -0.35 from a plot of log K_d versus . Interestingly, propionyl- and butyryl-lysine peptides were found to bind tighter to Hst2 compared with acetyl-lysine peptide and showed measurable rates of catalysis with Hst2, Sirt1, Sirt2, and Sirt3, suggesting propionyl- and butyryl-lysine proteins may be sirtuin substrates in vivo. Unique among the acetyl-lysine analog peptides examined, homocitrulline peptide produced ADP-ribose instead of the corresponding OAADPr analog. The electron-withdrawing nature of each acetyl analog had a profound impact on the deacylation rate between deacetylase classes. The rate of catalysis with the acetyl-lysine analog peptides varied over five orders of magnitude with the class III deacetylase Hst2, revealing a linear free energy relationship with a slope of -1.57 when plotted versus the Taft constant, ^*. HDAC8, a class I deacetylase, displayed the opposite trend with a slope of +0.79. These results are applicable toward the development of selective substrates and other mechanistic probes of protein deacetylases.

Received for publication, September 20, 2007 , and in revised form, October 18, 2007.

^* This work was supported by National Institutes of Health Grant GM065386 (to J. M. D.) and by National Institutes of Health Biotechnology Training Grant NIH 5 T32 GM08349 (to B. C. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S9 and S10.

¹ To whom correspondence should be addressed: 1300 University Ave., 551 MSC, Madison, WI 53706-1532. Tel.: 608-265-1859; Fax: 608-262-5253; E-mail: jmdenu{at}wisc.edu.

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