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J. Biol. Chem., Vol. 279, Issue 38, 40122-40129, September 17, 2004

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Coenzyme Specificity of Sir2 Protein Deacetylases

IMPLICATIONS FOR PHYSIOLOGICAL REGULATION^*

Manning T. Schmidt, Brian C. Smith, Michael D. Jackson¶, and John M. Denu||

From the Departments of Biomolecular Chemistry and Chemistry, University of Wisconsin, Madison, Wisconsin 53706 and the ^¶Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, Portland, Oregon 97239

Sir2 (silent information regulator 2) enzymes catalyze a unique protein deacetylation reaction that requires the coenzyme NAD⁺ and produces nicotinamide and a newly discovered metabolite, O-acetyl-ADP-ribose (OAADPr). Conserved from bacteria to humans, these proteins are implicated in the control of gene silencing, metabolism, apoptosis, and aging. Here we examine the role of NAD⁺ metabolites/derivatives and salvage pathway intermediates as activators, inhibitors, or coenzyme substrates of Sir2 enzymes in vitro. Also, we probe the coenzyme binding site using inhibitor binding studies and alternative coenzyme derivatives as substrates. Sir2 enzymes showed an exquisite selectivity for the nicotinamide base coenzyme, with the most dramatic losses in binding affinity/reactivity resulting from relatively minor changes in the nicotinamide ring, either by reduction, as in NADH, or by converting the amide to its acid analogue. Both ends of the dinucleotide NAD⁺ are shown to be critical for high selectivity and high affinity. Among the NAD⁺ metabolites tested none were able to allosterically activate, although all led to various extents of inhibition, consistent with competition at the coenzyme binding site. Nicotinamide was the most potent inhibitor examined, suggesting that cellular nicotinamide levels would provide an effective small molecule regulator of protein deacetylation and generation of OAADPr. The presented findings also suggest that changes in the physiological NAD⁺:NADH ratio, without a change in NAD⁺, would yield little alteration in Sir2 activity. That is, NADH is an extremely ineffective inhibitor of Sir2 enzymes (average IC₅₀ of 17 mM). We propose that changes in both free nicotinamide and free NAD⁺ afford the greatest contribution to cellular activity of Sir2 enzymes but with nicotinamide having a more dramatic effect during smaller fluctuations in concentration.

Received for publication, July 6, 2004 , and in revised form, July 20, 2004.

^* This work was supported by American Cancer Society Grant RSG-01-029-01, National Institutes of Health (NIH) Grant GM65386 (to J. M. D.), NIH Biotechnology Training Grant NIH 5 T32 GM08349 (to B. C. S.), and by NIH Postdoctoral Fellowship DK07680-12 (to M. D. J.). 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.

^|| To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706-1532. Tel.: 608-265-1859; Fax: 608-262-5253; E-mail: jmdenu{at}wisc.edu.

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