Related Experiment Video
Updated: Jul 1, 2026
14:32
Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates
Published on: February 27, 2016
Structural insights into intermediate steps in the Sir2 deacetylation reaction
William F Hawse1, Kevin G Hoff, David G Fatkins
1Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Structure (London, England : 1993)
|September 13, 2008
Summary
Structural studies of the bacterial sirtuin Sir2Tm reveal key mechanistic insights into the deacetylation process. The research elucidates the roles of active site residues in stabilizing reaction intermediates.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Sirtuins are NAD(+)-dependent protein deacetylases crucial in cellular regulation.
- Understanding the deacetylation mechanism requires detailed structural information of reaction intermediates, which are currently lacking.
Purpose of the Study:
- To elucidate the deacetylation mechanism of the bacterial sirtuin, Sir2Tm.
- To provide structural insights into the chemical steps of the deacetylation reaction.
Main Methods:
- X-ray crystallography was used to determine the structures of Sir2Tm in complex with reaction intermediates.
- Biochemical assays were performed to investigate the roles of active site residues.
Main Results:
- Crystal structures of Sir2Tm complexed with an S-alkylamidate intermediate and a ternary complex were obtained.
- The invariant active site histidine was found to be critical for positioning the reaction intermediate.
- A conserved phenylalanine residue plays a role in shielding intermediates from base exchange.
Conclusions:
- The study provides key mechanistic insights into the Sir2 deacetylation reaction.
- Structural resolution of intermediates reveals the function of specific active site residues in catalysis.
Related Concept Videos
01:24Phase II Reactions: Acetylation Reactions
Acetylation, a phase II biotransformation reaction, introduces an acetyl group to drugs or their metabolites. Acetyltransferase enzymes facilitate this reaction, which resembles α-amino acid conjugation due to the addition of a functional group to the drug molecule.
The substrates for acetylation are typically drugs or their metabolites with an amino, sulfonamide, or hydrazine functional group. Acetylation can occur at several points in the drug molecule, including primary, secondary, and...
The substrates for acetylation are typically drugs or their metabolites with an amino, sulfonamide, or hydrazine functional group. Acetylation can occur at several points in the drug molecule, including primary, secondary, and...
02:23SN2 Reaction: Stereochemistry
In an SN2 reaction, the nucleophilic attack on the substrate and departure of the leaving group occurs simultaneously through a transition state. As the nucleophile approaches the substrate from the back-side, the configuration of the substrate carbon changes from tetrahedral to trigonal bipyramidal and then back to tetrahedral, leading to an inversion in the configuration of the product.
If the substrate is an achiral molecule at the α-carbon, the inversion of configuration is not observed.
If the substrate is an achiral molecule at the α-carbon, the inversion of configuration is not observed.
02:26SN2 Reaction: Transition State
An SN2 reaction of an alkyl halide is a single-step process in which bond formation between the nucleophile and the substrate and bond breaking between the substrate and the halide occurs simultaneously through a transition state without forming an intermediate.
When the nucleophile approaches the electrophilic carbon with its lone pairs, the halide acts as a leaving group and moves away with the electron-pair bonded to the carbon. Dotted partial bonds represent the bonds being formed or broken...
When the nucleophile approaches the electrophilic carbon with its lone pairs, the halide acts as a leaving group and moves away with the electron-pair bonded to the carbon. Dotted partial bonds represent the bonds being formed or broken...
01:14Alkylation of β-Diester Enolates: Malonic Ester Synthesis
Malonic ester synthesis is a method to obtain α substituted carboxylic acids from ꞵ-diesters such as diethyl malonate and alkyl halides.
02:15SN1 Reaction: Stereochemistry
This lesson provides an in-depth discussion of the stereochemical outcomes in an SN1 reaction.
In the first step of an SN1 reaction, the bond between the electrophilic carbon and the leaving group ionizes to generate the carbocation intermediate. The second step of the mechanism is the nucleophilic attack.
In the formed carbocation, the positively charged carbon is sp2 hybridized with a trigonal planar geometry. As all the three substituents lie on the same plane, a plane of symmetry for the...
In the first step of an SN1 reaction, the bond between the electrophilic carbon and the leaving group ionizes to generate the carbocation intermediate. The second step of the mechanism is the nucleophilic attack.
In the formed carbocation, the positively charged carbon is sp2 hybridized with a trigonal planar geometry. As all the three substituents lie on the same plane, a plane of symmetry for the...
01:26Protein Modifications in the RER
Modification of secretory and transmembrane proteins entering the rough ER begins in the ER lumen. These modifications aid in protein folding and stabilize the acquired tertiary structure. Protein modifications in the rough ER co-occur at different stages of protein folding.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.