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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: Biochemistry. 2011 Feb 22;50(11):1940–1949. doi: 10.1021/bi101606e

Table 3.

Kinetic Parameters and ADP Binding of bGRK1535H6 T8C/N480Ca

ROS phosphorylation peptide C phosphorylation ADP
kcat
(×10−3 s−1)		 KM
(×10−6 M)		 kcat/KM
(×103 M−1 s−1)		 kcat
(×10−6 s−1)		 KM
(×10−4 M)		 kcat/KM
(×10−2 M−1 s−1)		 KD
(µM)
wt 25 ± 4 3.4 ± 1.7 7.2 ± 2.7 33 ± 6 3.2 ± 1.4 11 ± 3 0.84 ± 0.15
wt, K3Fe(CN)6b 27 ± 2 3.2 ± 0.8 8.5 ± 1.6 39 ± 8 4.4 ± 1.8 8.7 ± 2.0
T8C/N480C 17 ± 2 4.7 ± 1.4 3.6 ± 0.7 55 ± 18 5.2 ± 3.5 11 ± 4 0.88 ± 0.30
T8C/N480C, K3Fe(CN)6b 23 ± 4 3.1 ± 1.9 7.4 ± 3.5 410 ± 60 2.7 ± 1.0 152 ± 33 1.2 ± 0.2
T8C/N480C, K3Fe(CN)6, DTTb 23 ± 2 5.8 ± 1.5 3.9 ± 0.7 78 ± 8 4.6 ± 1.2 17 ± 3 1.4 ± 0.8
a

Numbers represent means ± the standard error of the fit calculated from three to six independent experiments. Reactions were performed in 100 mM HEPES-NaOH (pH 7.5), 0.15 M NaCl, 10 mM MgCl2, and 1 mM EDTA for <5 min for ROS phosphorylation and <2 h for peptide phosphorylation at room temperature.

b

Proteins were treated with 1 mM K3Fe(CN)6 at room temperature for 1 h, and then monomers were purified by size-exclusion chromatography. Some of the protein was then reduced with 10 mM DTT at 4 °C for 1 h before the experiments described above.