Dual role of Zn2+ in maintaining structural integrity and suppressing deacetylase activity of SIRT1

Zn2+ directly participates in catalysis of histone deacetylase (HDAC) Classes I, II, IV enzymes while its role in HDAC Class III activity is not well established. Herein we investigated the effects of Zn2+ on the deacetylase activity of sirtuin 1 (silent mating type information regulation 2 homolog 1, SIRT1). We found that the inherent Zn2+ at the zinc-finger motif of SIRT1 is essential for the structural integrity and the deacetylase activity of SIRT1, whereas the exogenous Zn2+ strongly inhibits the deacetylase activity with an IC50 of 0.82 mu M for Zn(Gly)(2). SIRT1 activity suppressed by the exogenous Zn2+ can be fully recovered by the metal chelator EDTA but not by the activator resveratrol. We also identified Zn2+ as a noncompetitive inhibitor for the substrates of NAD(+) and the acetyl peptide P53-AMC. The 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence titration experiments and site-directed mutagenesis study suggested that the exogenous Zn2+ binds to SIRT1 but not at the zinc-finger motif. These results indicate that Zn2+ plays a dual role in SIRT1 activity. Inherent Zn2+ at the zinc-finger motif is structurally related and essential for SIRT1 activity. On the other hand, Zn2+ may also bind to another site different from the zinc-finger motif or the binding sites for the substrates or resveratrol and act as a potent inhibitor of SIRT1. (C) 2009 Elsevier Inc. All rights reserved.