Fig 1.
Schematic Representation of the Sirtuin Catalyzed Reaction Mechanism: The sirtuin catalyzed reaction proceeds via the nucleophilic attack of the carbonyl oxygen of the acylated substrate to the C1’ ribose of NAD+, resulting in the cleavage of the nicotinamide moiety of NAD+ and formation of C1′-O-alkylimidate intermediate.
The alkylimidate intermediate undergoes the cyclization reaction followed by hydrolysis, generating the deacylated product and 2′-O-acyl-ADP-ribose (OAADPr) species. The latter is slowly isomerized (non-enzymatically) to form 3′-OAADPr.
Fig 2.
Comparative Structural Features in the Catalytic Domains of SIRT5 and SIRT1.
(A) The superimposition (ribbon diagrams) of the catalytic domains of human SIRT5 (blue) and SIRT1 (tan). (B) A close view at the active site pockets of SIRT5 and SIRT1. The side chains of Y102 and R105I (yellow) in SIRT5 and of A313 and I316 (pink) in SIRT1 are presented as ball-and-stick models.
Fig 3.
Circular Dichroim (CD) Spectra of SIRT5 Variants and SIRT1.
The mean residue ellipticities of 10 μM enzymes as a function of wavelength are shown. The experiments were performed in 5 mM Tris-HCl buffer, pH 7.5 using a quartz cuvette of 1 mm path length.
Fig 4.
Thermal Denaturation Profiles of SIRT5 Variants and SIRT1.
The ellipticity of (A) SIRT5 wild type, (B) Y102A, (C) R105I, (D) Y102A/R105I and (E) SIRT1were measured at 208 nm as a function of temperature. The solid smooth lines represent the best fit of the data by the single phase Boltzmann equation (Eq 2) to derive the Tm values. (F) The CD spectra of wild-type and mutant enzymes upon heating at their final melting temperatures followed by cooling at 25°C
Table 1.
Melting Temperatures of SIRT5 Variants as well as SIRT1.
Fig 5.
Real-time Progress Curve for SIRT5-catalyzed Desuccinylation Reaction.
The time courses of the SIRT5 catalyzed reaction using the fixed concentrations of Ac-Suclys-AMC substrate and NAD+, and varying concentrations of trypsin as the coupling enzyme are shown. The dashed curves represent the time dependent increase in fluorescence at 460 nm (λex = 350 nm) due to release of 7-hydroxyl-4-methylcoumarin (AMC) from the substrate. The solid lines indicate the linear regression analysis of the data after the initial lag phases (the dashed curves).
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.
Table 2.
Steady-state Kinetic Parameters of SIRT5 Variants and SIRT1 on Acetyl and Succinyl substrates.
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.
Table 3.
Nicotinamide Inhibition against SIRT5 Variants and SIRT1.
Fig 8.
Effects of Isonicotinamide on SIRT1 and SIRT 5 Catalyzed reactions.
(A) Dose-response of SIRT1 deacetylation activity in the presence of varying concentrations of isonicotinamide (from 0 to 15 mM) and NAD+ (50 μM and 5 mM). The solid smooth lines represent the best fit of the data for the IC50 values as 7.0 ± 2.9 mM (in the presence of 50 μM NAD+) and 8.9 ± 3.3 mM (in the presence of 5 mM NAD+), respectively. (B) Dose-response of SIRT5 desuccinylation activity in the presence of varying concentrations of isonicotinamide (from 0 to 15 mM) and NAD+ (50 μM and 5 mM). The solid smooth lines represent the best fit of the data for the IC50 values as 11.1 ± 2.2 mM (in the presence of 50 μM NAD+) and 17.1 ± 4.0 mM (in the presence of 5 mM NAD+), respectively.
Table 4.
Isonicotinamide (iNAM) Effect on the Nicotinamide Inhibition of SIRT1 and SIRT5.
Fig 9.
Structure of EX527 and its Effects on SIRT5 Variant Catalyzed Reactions.
(A) Structure of EX527. (B) Relative activities of SIRT5 variants in the absence and presence of 50 μM EX527. (C) Dose-response of SIRT1 deacetylase activity in the presence of increasing concentration of EX527 (from 0 to 40 μM). (D) Dose-response of SIRT5 Y102A/R105I deacetylase activity in the presence of increasing concentration of EX527 (from 0 to 200 μM). For (C) and (D), the solid smooth lines represent the best fit of the data for the IC50 of EX527 against SIRT1 and SIRT5 Y102A/R105I as being equal to 0.9 ± 0.2 μM and 21.7 ± 1.0 μM, respectively.
Fig 10.
ITC profile for the binding of EX527 to SIRT5 Y102A/R105I in the absence (left) and presence of 10 mM NAD+ (right).
The top panels show the raw calorimetric data generated by titration of 20 μM SIRT5 Y102A/R105I by 45 injections (5 μl each) of 500 μM EX527. The area under each peak was integrated and plotted against the molar ratio of EX527 to SIRT5 Y102A/R105I. The solid lines (right panel) represent the best fit of the experimental data for the single binding site model, yielding the magnitudes of n, ΔH° and Ka as being equal to 1.2, −5.3 ± 0.1 kcal/mol and (2.5 ± 0.2) × 105 M-1, respectively.