Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases.
The Silent information regulator 2 (Sir2) family of enzymes consists of NAD(+)-dependent histone/protein deacetylases that tightly couple the hydrolysis of NAD(+) and the deacetylation of an acetylated substrate to form nicotinamide, the deacetylated product, and the novel metabolite O-acetyl-ADP-ribose (OAADPR). In this paper, we analyzed the substrate specificity of the yeast Sir2 (ySir2), the yeast HST2, and the human SIRT2 homologues toward various monoacetylated histone H3 and H4 peptides, determined the basic kinetic mechanism, and resolved individual chemical steps of the Sir2 reaction. Using steady-state kinetic analysis, we have shown that ySir2, HST2, and SIRT2 exhibit varying catalytic efficiencies and display a preference among the monoacetylated peptide substrates. Bisubstrate kinetic analysis indicates that Sir2 enzymes follow a sequential mechanism, where both the acetylated substrate and NAD(+) must bind to form a ternary complex, prior to any catalytic step. Using rapid-kinetic analysis, we have shown that after ternary complex formation, nicotinamide cleavage occurs first, followed by the transfer of the acetyl group from the donor substrate to the ADP-ribose portion of NAD(+) to form OAADPr and the deacetylated product. Product and dead-end inhibition analyses revealed that nicotinamide is the first product released followed by random release of OAADPr and the deacetylated product.
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.
Margie T
MT
Borra
Michael R
MR
Langer
James T
JT
Slama
John M
JM
Denu
D000107:Acetylation; D000595:Amino Acid Sequence; D002384:Catalysis; D003956:Dialysis; D004791:Enzyme Inhibitors / Q000737:chemistry; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D038961:O-Acetyl-ADP-Ribose / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D056565:Sirtuin 2; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D038961:O-Acetyl-ADP-Ribose; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D009243:NAD; C423033:HST2 protein, S cerevisiae; C087778:SIR2 protein, S cerevisiae; C474199:SIRT2 protein, human; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases