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

Lei Chen, Yu Feng, Yinqiu Zhou, Weiliang Zhu, Xu Shen, Kaixian Chen, Hualiang Jiang and Dongxiang Liu

a b s t r a c t

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 lM 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.