Role of the Substrate Specificity-Defining Residues of Human SIRT5 in Modulating the Structural Stability and Inhibitory Features of the Enzyme
Fig 6
Two Substrates Reactions of SIRT5.
The two substrate SIRT5 catalyzed reaction was performed under the steady-state condition with varying concentrations of Ac-SucLys-AMC substrate (25, 50, 100, 200, and 500 μM) and NAD+ (50, 70, 100, 150, 250, 350, 500, and 700 μM). The data were fitted to the bi-substrate ternary complex kinetic mechanism for the binding of the substrate followed by the binding of NAD+ by Eq 4 using Grafit software. The initial rates of SIRT5 catalysis as a function of increasing concentration of Ac-SucLys-AMC substrate at changing fixed concentrations of NAD+ (A), and as a function of increasing concentration of NAD+ at changing fixed concentrations of Ac-SucLys-AMC substrate (B) The double-reciprocal plot of 1/v vs 1/[NAD+] at increasing concentration of Ac-Suclys-AMC substrate (C), and the double-reciprocal plot of 1/v vs 1/[Ac-Suclys-AMC] at increasing concentration of NAD+ (D) are also shown.