. 2018 Dec 11;7:e37921. doi: 10.7554/eLife.37921

Table 3. Kinetic properties of rat and human SETD3 proteins.

Kinetic properties were determined with the use of purified recombinant C-terminal His₆-tagged SETD3 protein.

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

elife-37921-table3-data1.xls^{(67KB, xls)}

DOI: 10.7554/eLife.37921.027

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

elife-37921-table3-data2.xls^{(66KB, xls)}

DOI: 10.7554/eLife.37921.028

Table 3—source data 3. Determination of the kinetic parameters of SETD3-catalyzed methylation of peptide H.

elife-37921-table3-data3.xls^{(58.5KB, xls)}

DOI: 10.7554/eLife.37921.029

Substrate		Rat SETD3				Human SETD3
	V_max	K_M	k_cat	k_cat/K_M	V_max	K_M	k_cat	k_cat/K_M
	nmol min⁻¹ mg⁻¹	µM	min⁻¹	min⁻¹ µM⁻¹	nmol min⁻¹ mg⁻¹	µM	min⁻¹	min⁻¹ µM⁻¹
β-actin	11.280 ± 1.018	2.996 ± 0.507	0.80	0.27	9.091 ± 0.308	0.752 ± 0.070	0.65	0.86
S-adenosyl-L- methionine	8.053 ± 0.136	0.109 ± 0.008	0.57	5.23	8.649 ± 0.119	0.116 ± 0.007	0.61	5.25
Peptide H	0.064 ± 0.004	10590 ± 1373	0.005	4.7 × 10⁻⁷	0.029 ± 0.003	8729 ± 1949	0.002	2.3 × 10⁻⁷

Determinations for S-adenosyl-L-methionine (SAM) were performed with the SETD3 preparations (0.04–0.05 µg protein, 5–7 nM), which were incubated for 8 min at 37°C in the reaction mixture containing 5 µM recombinant β-actin and variable concentrations of [¹H+³H] SAM (≈320 × 10³ cpm). The measurements for β-actin were obtained following a 5 min incubation of SETD3 in the presence of 1 μM concentration of [¹H+³H] SAM (100 pmol, ≈300 × 10³ cpm). The kinetic parameters of the enzymatic reaction for actin peptide H (YPIEHGIVT) were determined with SETD3 preparations (8.5–9.2 µg protein, 1.2–1.3 µM) incubated for 30 min in the presence of [¹H+³H] SAM (100 pmol, ≈290 × 10³ cpm). In all experiments, the reaction mixture contained the homogenous recombinant S-adenosyl-L-homocysteine (SAH) nucleosidase (1.6 µg protein, 600 nM, E. coli) and adenine deaminase (3.9 µg protein, 600 nM, B. subtilis) to prevent SAH accumulation. Values are the means of three or four independent experiments. The values for standard error of the mean (S.E.) are also given.