Role of the Substrate Specificity-Defining Residues of Human SIRT5 in Modulating the Structural Stability and Inhibitory Features of the Enzyme
Fig 7
Nicotinamide Inhibition of SIRT5 Variants and SIRT1.
Nicotinamide exhibits competitive inhibition against NAD+ during the wild-type SIRT5 and its Y102A mutant catalyzed desuccinylation reactions (A and B); mixed type of inhibition against NAD+ during the Y102A/R105I SIRT5 mutant and SIRT1 catalyzed deacetylation reaction (C and D). The wild-type SIRT5 catalyzed reactions (A) were performed in the presence of 100 μM Ac-SucLys-AMC and varying concentrations of NAD+ (from 40 to 300 μM) in the presence of 0, 25, 50 and 100 μM concentrations of nicotinamide. The Y102A mutant catalyzed reactions (B) were performed in the presence of 500 μM Ac-SucLys-AMC and varying concentrations of NAD+ (from 70 to 800 μM) in the presence of 0, 50, 100 and 200 μM nicotinamide. The Y102A/R105I mutant catalyzed deacetylation reactions (C) were performed in the presence of 500 μM Fluo-de-lys® acetylated substrate, and varying concentrations of NAD+ concentration (from 200 to 1300 μM), in the presence of 0, 100, 200 and 400 μM nicotinamide. SIRT1 reactions (D) were performed in the presence of 100 μM Fluo-de-lys® acetylated substrate, and varying concentrations of NAD+ (from 40 to 300 μM) in the presence of 0, 50, 100, and 200 μM nicotinamide. Data were fitted using DYNAFIT software and the derived Ki values are summarized in Table 3.