Ligand Binding and Catalysis in Selected Sirtuin Isozymes

Abstract

Due to their intimate roles in survival, longevity as well as pathogenesis via “epigenetic” and “metabolic” regulatory mechanisms, sirtuins have gained considerable interest toward undertaking detailed biochemical/biophysical studies. The present study was designed to ascertain the mechanistic details of ligand binding and catalysis in selected sirtuin isozymes (viz., SIRT1 and SIRT5) from the point of view of designing isozyme selective inhibitors as potential therapeutics. By screening of the in-house synthesized compounds, two barbiturate derivatives were identified as the SIRT5 selective inhibitors. These, along with some of known inhibitors of SIRT1 and SIRT5, namely, MH5-75, nicotinamide, suramin were investigated by a combination of spectroscopic, kinetic, and thermodynamic techniques. The influence of the sirtuin inhibitors in modulating the structural features of the enzymes were ascertained by CD spectroscopic, lifetime fluorescence, and thermal denaturation studies using wild-type and selected site-specific mutant enzymes. The experimental data revealed that the substrate selectivity and inhibitory features in SIRT5 were manifested via the mutual cooperation between Y102 and R105 residues of the enzyme, and the overall catalytic feature of the enzyme was modulated by changes in the protein structure. Whereas the stoichiometry of SIRT1 to suramin remained invariant as 1:1, that of SIRT5 to suramin increased from 1:1 to 2:1 upon increase in the molar ratio of the enzyme to the ligand. A comparative account of the experimental data presented herein sheds light on the structural-functional differences between SIRT1 and SIRT5, leading to the design of isozyme selective inhibitors as therapeutic tools for the treatment of sirtuin associated diseases.