Faculty Collaboration Database

Sir2 protein deacetylases: evidence for chemical intermediates and functions of a conserved histidine. Biochemistry 2006 Jan 10;45(1):272-82

Date

01/04/2006

Pubmed ID

16388603

Pubmed Central ID

PMC2519119

DOI

10.1021/bi052014t

Scopus ID

2-s2.0-30144431571 (requires institutional sign-in at Scopus site)   93 Citations

Abstract

Sir2 NAD+-dependent protein deacetylases are implicated in a variety of cellular processes such as apoptosis, gene silencing, life-span regulation, and fatty acid metabolism. Despite this, there have been relatively few investigations into the detailed chemical mechanism. Sir2 proteins (sirtuins) catalyze the chemical conversion of NAD+ and acetylated lysine to nicotinamide, deacetylated lysine, and 2'-O-acetyl-ADP-ribose (OAADPr). In this study, Sir2-catalyzed reactions are shown to transfer an 18O label from the peptide acetyl group to the ribose 1'-position of OAADPr, providing direct evidence for the formation of a covalent alpha-1'-O-alkylamidate, whose existence is further supported by the observed methanolysis of the alpha-1'-O-alkylamidate intermediate to yield beta-1'-O-methyl-ADP-ribose in a Sir2 histidine-to-alanine mutant. This conserved histidine (His-135 in HST2) activates the ribose 2'-hydroxyl for attack on the alpha-1'-O-alkylamidate. The histidine mutant is stalled at the intermediate, allowing water and other alcohols to compete kinetically with the attacking 2'-hydroxyl. Measurement of the pH dependence of kcat and kcat/Km values for both wild-type and histidine-to-alanine mutant enzymes confirms roles of this residue in NAD+ binding and in general-base activation of the 2'-hydroxyl. Also, transfer of an 18O label from water to the carbonyl oxygen of the acetyl group in OAADPr is consistent with water addition to the proposed 1',2'-cyclic intermediate formed after 2'-hydroxyl attack on the alpha-1'-O-alkylamidate. The effect of pH and of solvent viscosity on the kcat values suggests that final product release is rate-limiting in the wild-type enzyme. Implications of this new evidence on the mechanisms of deacetylation and possible ADP-ribosylation catalyzed by Sir2 deacetylases are discussed.

Author List

Smith BC, Denu JM

Author

Brian C. Smith PhD Associate Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Acetylation
Adenosine Diphosphate Ribose
Alanine
Binding Sites
Catalysis
Histidine
Hydrogen-Ion Concentration
Kinetics
Mutation
NAD
Protein Conformation
Sirtuins
Spectrometry, Mass, Electrospray Ionization
Substrate Specificity
Thermodynamics