Table 1.

Mutational effects on 5′ kinase activity with _HOCp acceptor

Pnkp	Km HOCp (µM)	kcat (min−1)
WT	31 ± 4	98 ± 16
Y52A	16 ± 3	69 ± 2
K54A	12 ± 1	80 ± 5
E57A	157 ± 22	3.3 ± 0.3
T61A	36 ± 4	21 ± 1.5
N89A	24 ± 6	50 ± 9
R92A	105 ± 7	74 ± 6
K129A	43 ± 9	33 ± 2
V131A	113 ± 13	32 ± 2
V131F	460 ± 46	0.33 ± 0.03
P132G	67 ± 7	56 ± 2
V135A	39 ± 6	33 ± 2
V135F	323 ± 28	23 ± 1.3

Reaction mixtures (10 µl) containing 70 mM Tris–HCl (pH 7.6), 10 mM MgCl₂, 5 mM DTT, 25 µM [γ³²P]ATP, wild-type or mutant Pnkp and varying concentrations of 3′-CMP were incubated for 20 min at 37°C. The extents of [α³²P]pCp product formation were plotted as a function of 3′-CMP concentration. K_m and k_cat were obtained by nonlinear regression curve fitting of the data for each experiment to the Michaelis–Menten equation in Prism. The values shown are averages of three independent 3′-CMP titration experiments ±SEM. Representative 3′-CMP titration profiles are shown in Supplementary Figure S1.