Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions.
Level 5 - mechanism / opinion, no new human data
In vitro bench biochemistry and enzymology study without human clinical data
PubMed 11747420 · doi:10.1021/bi011858j
What was done
The authors investigated the enzymatic reaction mechanism and products of NAD+-dependent protein deacetylation catalyzed by the Sir2 enzyme family using peptide substrates. Reaction intermediates, products, regioisomers, anomeric forms, interconversion rates, and equilibria were characterized using NMR, HPLC, 18O exchange, and mass spectrometry (MS) methods.
What was found
The abstract reports qualitative biochemical steps and reaction products without providing numerical rate constants or equilibrium values. The reaction yields deacetylated peptide and 2'- and 3'-regioisomers of O-acetyl ADP-ribose (AADPR). The pathway proceeds via ADP-ribosylation of the peptide acyl oxygen forming an O-alkyl amidate intermediate, attack of the 2'-OH group forming a 1',2'-acyloxonium species, water hydrolysis to 2'-AADPR, and non-enzymatic intramolecular transesterification equilibrating 2'- and 3'-AADPR.
Why it matters
This study defines the catalytic mechanism and chemical products of NAD+-dependent deacetylation by Sir2 enzymes, demonstrating that deacetylation is directly coupled to stoichiometric NAD+ cleavage to generate O-acetyl-ADP-ribose.
Limits
This is an in vitro bench enzymology study using synthetic peptide substrates rather than full-length protein complexes or in vivo systems. The abstract provides no quantitative kinetic parameters, rate constants, or equilibrium constants. Biological signaling roles of the AADPR products were not experimentally tested.
Cited by
- supports SIRT1 requires NAD as a co-substrate, transferring the chemical group removed from epigenetic structures to NAD during its enzymatic reaction.