Orphan macrodomain protein (human C6orf130) is an O-acyl-ADP-ribose deacylase: solution structure and catalytic properties - PubMed (original) (raw)

C6orf130 deacetylates _O_AADPr. a, sequence alignment C6orf130 with other MacroD-like proteins human MacroD1, human MacroD2, E. coli YmdB, and S. aureus SAV0325. b, steady-state kinetic analysis of C6orf130. The deacetylation reaction of _O_AADPr catalyzed by C6orf130 follows saturation kinetics. The steady-state kinetic parameters are determined by radioactive assays for acetate formation and HPLC assays for ADP-ribose formation. Apparent Km as measured was 182 ± 17 μ

m

; the _k_cat as measured was 0.31 ± 0.03 s−1. The reaction mixtures contained 0.5 μ

m

C6orf130. c, inhibition of C6orf130 deacetylase activity by ADPr. The deacetylation reactions were carried out in 50 m

m

Tris-HCl, pH 7.3, containing 0.5 μ

m

C6orf130. The reactions were initiated in the presence of 0 μ

m

(circles), 100 μ

m

(squares), 200 μ

m

(diamonds), and 400 μ

m

(triangles) ADPr, respectively. The _Km_′ values measured were 183.3, 340.6, 480.1, and 782.2 μ

m

in the presence of 0, 100, 200, and 400 μ

m

initial ADPr, respectively. The _V_max values were 0.31, 0.29, 0.29, and 0.30 s−1. The Ki was determined to be 119.3 ± 4.5 μ

m

. The error bars represent standard deviations calculated from the measured initial velocities at each substrate concentration from three separate experiments. d, inhibition of C6orf130 deacetylase activity by _N_AADPr. The deacetylation reactions were carried out under the same conditions as described in c in the presence of 0 μ

m

(circles), 125 μ

m

(squares), 250 μ

m

(diamonds), and 500 μ

m

(triangles) _N_AADPr, respectively. The _Km_′ values measured were 183.3, 340.6, 480.1, and 782.2 μ

m

in the presence of 0, 125, 250, and 500 μ

m

initial _N_AADPr, respectively. The _V_max values were 0.31, 0.29, 0.29, and 0.30 s−1. The Ki was determined to be 223.3 ± 11.4 μ

m

. The error bars represent standard deviations calculated from the initial velocities at each substrate concentration from three separate experiments.