. 2019 Feb 20;8:e43676. doi: 10.7554/eLife.43676

Table 3. Kinetic properties of wildtype SETD3 and its mutants.

Kinetic parameters were determined by using purified recombinant N-terminal His₆-tagged SETD3 proteins.

Table 3—source data 1. Determination of the kinetic parameters of SETD3-catalyzed methylation of actin (for β-actin as the substrate).

elife-43676-table3-data1.xlsx^{(239.3KB, xlsx)}

DOI: 10.7554/eLife.43676.018

Table 3—source data 2. Determination of kinetic parameters of SETD3-catalyzed methylation of actin (for AdoMet as the substrate).

elife-43676-table3-data2.xlsx^{(245.7KB, xlsx)}

DOI: 10.7554/eLife.43676.019

SETD3	Substrate		Kinetic parameters
		V_max	K_M	k_cat	k_cat/K_M
		nmol min⁻¹ mg⁻¹	µM	min⁻¹	min⁻¹ µM⁻¹
Wildtype	β-actin	13.550 ± 0.364	0.502 ± 0.041	0.809	1.612
R75A		0.037 ± 0.002	0.565 ± 0.071	0.002	0.004
R215A		3.553 ± 0.068	1.087 ± 0.056	0.212	0.195
N256A		0.512 ± 0.019	0.449 ± 0.053	0.031	0.069
N256D		0.973 ± 0.017	0.305 ± 0.0196	0.058	0.190
N256Q		10.860 ± 0.172	0.784 ± 0.035	0.649	0.828
N278A		0.019 ± 0.000	0.047 ± 0.009	0.001	0.021
Y313F		0.367 ± 0.005	0.581 ± 0.023	0.022	0.038
R316A		2.903 ± 0.0477	1.115 ± 0.049	0.173	0.155
Wildtype	AdoMet	11.260 ± 0.466	0.111 ± 0.020	0.673	6.063
R75A		0.265 ± 0.072	3.601 ± 1.363	0.016	0.004
R215A		3.666 ± 0.078	0.165 ± 0.013	0.219	1.327
N256A		1.136 ± 0.038	0.686 ± 0.052	0.068	0.099
N256D		0.671 ± 0.021	0.165 ± 0.019	0.040	0.242
N256Q		6.368 ± 0.266	0.103 ± 0.019	0.380	3.689
N278A		0.018 ± 0.001	0.350 ± 0.042	0.001	0.003
Y313F		1.623 ± 0.348	6.543 ± 1.736	0.097	0.015
R316A		4.099 ± 0.054	0.149 ± 0.008	0.244	1.638

Determinations for S-adenosyl-L-methionine (AdoMet) were performed with the SETD3 preparations (0.05–5.00 µg protein, 8.38–838 nM), which were incubated for 10 min at 37°C in a reaction mixture containing 5 µM recombinant human β-actin and variable concentrations of [¹H+³H] AdoMet (≈330 × 10³ cpm). The measurements for β-actin were done following a 10 min incubation of SETD3 in the presence of a 0.8 μM concentration of [¹H+³H] AdoMet (80 pmol, 300–700 × 10³ cpm). In all experiments, the reaction mixture contained the homogenous recombinant AdoHcy nucleosidase (1.6 µg protein, 600 nM, E. coli) and adenine deaminase (3.9 µg protein, 600 nM, B. subtilis) to prevent S-adenosyl-L-homocysteine (AdoHcy) accumulation. Values are the means of three separate experiments. The S.E. values are also given.