. Author manuscript; available in PMC: 2018 Jan 10.

Published in final edited form as: Biochemistry. 2016 Dec 27;56(1):33–46. doi: 10.1021/acs.biochem.6b00943

Table 3.

V_max, K_m, and k_cat Values for Incorporation of TTP and AZTTP by AZTr RT and of dCTP and ddCTP by RT^K65R at 6 and 0.25 mM Mg²⁺

	condition^a	[RT] (nM)	V_max(nM/min)	k_cat^b (min⁻¹)	K_m (μM)	k_cat/K_m^c (x-fold decrease)	p value^d
AZTr	6 mM Mg²⁺ dTTP	1	1.6 ± 0.2	1.6 ± 0.2	4.8 ± 0.5	0.33	–
	0.25 mM Mg²⁺ dTTP	1	0.55 ± 0.22	0.55 ± 0.22	2.2 ± 1	0.25 (1.3)	0.05
	6 mM Mg²⁺ AZTTP	1	1.3 ± 0.1	1.3 ± 0.1	5.2 ± 1.0	0.25	–
	0.25 mM Mg²⁺ AZTTP	1	0.32 ± 0.05	0.32 ± 0.05	5.5 ± 1.8	0.06 (4.2)	0.009
K65R	6 mM Mg²⁺ dCTP	2	1.5 ± 0.4	0.75 ± 0.18	1.6 ± 0.5	0.59	–
	0.25 mM Mg²⁺ dCTP	2	0.24 ± 0.02	0.12 ± 0.01	1.0 ± 0.4	0.15 (5.2)	0.007
	6 mM Mg²⁺ ddCTP	2	1.1 ± 0.1	0.53 ± 0.04	6.2 ± 0.3	0.08	–
	0.25 mM Mg²⁺ ddCTP	2	0.07 ± 0.04	0.03 ± 0.02	9.1 ± 0.5	0.003 (27)	<0.001

Assays were conducted with a 20-nucleotide template and 19-nucleotide 5′ end-labeled primer as described previously.³⁷ The single template-directed nucleotide was a T opposite an A and a C opposite a G for AZT and ddCTP, respectively. All assays used 0.8 or 1.6 nM RT. Results were averages of three experiments ± SD.

k_cat was calculated by dividing V_max by the enzyme concentration.

The x-fold decrease in enzyme efficiency (as judged by k_cat/K_m) compared to the 6 mM result (number above) with the same nucleotide or inhibitor.

p values were calculated using a standard Student’s t test for k_cat/K_m values between 0.25 and 6 mM Mg²⁺.