9-Fluorenylmethoxycarbonyl-labeled peptides as substrates in a capillary electrophoresis-based assay for sirtuin enzymes

Abstract

Sirtuins are the class III histone deacetylases that catalyze the deacetylation of acetyl-lysine residues of histones and other proteins using nicotinamide adenine dinucleotide (NAD +) as the cofactor. The reaction yields the deacetylated protein, nicotinamide, and 2’- O-acetyl-ADP-ribose. Three 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptides derived from the amino acid sequence of p53, Fmoc–KK(Ac)-NH 2, Fmoc–KK(Ac)L-NH 2, and Fmoc–RHKK(Ac)-NH 2, were characterized as substrates for two of the human sirtuins: SIRT1 and SIRT2. The deacetylation was monitored by a validated capillary electrophoresis assay. Efficient deacetylation by SIRT1 and SIRT2 was demonstrated for all three peptide substrates. The kinetics of the enzymatic reaction was determined with the Michaelis constants ( K m) varying between 16.7 and 34.6 μM for SIRT1 and between 34.7 and 58.6 μM for SIRT2. Resveratrol did not function as an activator for SIRT1 using the Fmoc-labeled peptides as SIRT substrates. The IC 50 values of sirtinol using the three peptide substrates were determined. Further sirtuin inhibitors were also evaluated.