Site-Specific Reactivity of Nonenzymatic Lysine Acetylation

Protein acetylation of lysine ε-amino
groups is abundant in cells, particularly within mitochondria. The
contribution of enzyme-catalyzed and nonenzymatic acetylation in mitochondria
remains unresolved. Here, we utilize a newly developed approach to
measure site-specific, nonenzymatic acetylation rates for 90 sites
in eight native purified proteins. Lysine reactivity (as second-order
rate constants) with acetyl-phosphate and acetyl-CoA ranged over 3
orders of magnitude, and higher chemical reactivity tracked with likelihood
of dynamic modification in vivo, providing evidence
that enzyme-catalyzed acylation might not be necessary to explain
the prevalence of acetylation in mitochondria. Structural analysis
revealed that many highly reactive sites exist within clusters of
basic residues, whereas lysines that show low reactivity are engaged
in strong attractive electrostatic interactions with acidic residues.
Lysine clusters are predicted to be high-affinity substrates of mitochondrial
deacetylase SIRT3 both in vitro and in vivo. Our analysis describing rate determination of lysine acetylation
is directly applicable to investigate targeted and proteome-wide acetylation,
whether or not the reaction is enzyme catalyzed.