FIGURE 3.

Deacetylation of O-acetyl-ADPr catalyzed by macroD-like proteins.A, [³H]acetate release from hydrolysis of O-[³H]acetyl-ADPr catalyzed by human MacroD1. OAADPr hydrolysis was monitored by measuring the radioactivity release as count per minute (CPM) due to [³H]acetate formation from O-[³H]acetyl-ADPr in the presence of 0 to 2.0 μm human MacroD1 and 800 μmOAADPr at pH 7.3. The data show the time dependent increase in free [³H]acetate formation and the rate of [³H]acetate formation increases with Macro D1 (E) concentration. B, HPLC assay of OAADPr hydrolysis catalyzed by human MacroD1. HPLC chromatograms monitored at 260 nm show the formation of ADPr from OAADPr in human MacroD1 catalyzed reaction. The chromatograms labeled 1–5 are reactions stopped by addition of TFA at 0, 3, 6, 10, and 16 min, respectively. The formation of ADPr from OAADPr depends on the presence of enzyme. Inset shows the time-dependent formation of ADPr from OAADPr in the presence of 0.5 μm MacroD1. C, LC-MS/MS analysis of human MacroD1 reaction. The precursor ions of 600.0 (OAADPr) and 558.0 (ADPr) and the major product ion of 346.0 shared by OAADPr and ADPr were selected. The SRM chromatogram shows the ion intensity versus time for OAADPr (dashed line) and ADPr (solid line) formed from the MacroD1 reaction stopped at 15 min. The inset shows the formation of ADPr increases with reaction time. The concentration of ADPr formed from each time point reaction was determined from external standards. D, steady-state kinetic analysis of human MacroD1. The deacetylation reaction of OAADPr catalyzed by MacroD1 follows saturation kinetics. The steady-state kinetic parameters are determined by radioactive assays for acetate formation and HPLC assays for ADP-ribose formation. An apparent K_m was measured to be 375 ± 55 μm. The V_max was measured to be 0.20 ± 0.04 s⁻¹. The reaction mixtures contain 0.5 μm MacroD1. E, inhibition of ADPr to human MacroD1 activity. The deacetylation reactions were carried out in 50 mm Tris-HCl of pH 7.3 containing 0.5 μm MacroD1. The reactions were initiated in the presence of 0 μm (closed squares), 100 μm (open squares), 200 μm (open diamonds), and 400 μm (open circles) ADPr, respectively. The K_m′ values measured are 375.8, 590.9, 986.6, and 1350.3 μm in the presence of 0, 100, 200, and 400 μm initial ADPr, respectively. The V_max values are 0.202, 0.204, 0.201, and 0.196 s⁻¹. The K_i was determined to be 145 ± 27 μm. The inhibition pattern is consistent with reversible competitive inhibition. F, kinetic comparison of wild-type MacroD1 and its variants. Conserved asparagine and aspartate residues located in vicinity to the 2′- and 3′-hydroxyl groups of the bound ADPr in the MacroD1 structure are mutated to alanine. The kinetic comparison of the wild type MacroD1 (closed squares) and its variants show that these residues are important to catalysis. The N171A (open squares), N174A (open diamonds), D184A (open circles), and N174A/D184A (open triangles), mainly affect the V_max. The K_m values are only minimally affected.