Table 1.

Pre–steady-state kinetic parameters for single nucleotide incorporation

Nucleotide	kp, s−1	Kd, μM	kp/Kd, μM−1⋅s−1	D-stereoselectivity*
Catalyzed by wild-type Polλ
D-dCTP	2.02 ± 0.06	0.81 ± 0.08	2.5
L-dCTP	(1.4 ± 0.1) × 10−4	0.67 ± 0.07	2.1 × 10−4	1.2 × 104
(–)3TC-TP	(3.0 ± 0.2) × 10−3	0.12 ± 0.02	2.5 × 10−2	100
(–)FTC-TP	(4.7 ± 0.1) × 10−3	0.35 ± 0.02	1.3 × 10−2	192
Catalyzed by the mutant R517A of Polλ
D-dCTP	(9.6 ± 0.2) × 10−4	0.22 ± 0.02	4.4 × 10−3
L-dCTP	Not determined†	36 ± 3

An incoming nucleotide was incorporated opposite the templating nucleotide dG in the single-nucleotide gapped DNA substrate 21-mer⋅19-mer/41-mer (SI Appendix, Fig. S1A) catalyzed by either wild-type human DNA Polλ or its mutant R517A at 37 °C.

^*D-stereoselectivity = (k_p/K_d)_D-dCTP/(k_p/K_d)_L-nucleotide.

^†No product formation was observed after 7 h.