Skip to main content
. 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 2.

Vmax, Km, and kcat Values for Incorporation of TTP and AZTTP, dCTP and ddCP, or dATP and EFdATP by HIV-2 RT at 6 and 0.25 mM Mg2+

conditiona [RT] (nM) Vmax (nM/min) kcatb (min−1) Km (μM) kcat/Kmc (x-fold decrease) p valued
6 mM Mg2+ dTTP 0.4 0.48 ± 0.07 1.2 ± 0.2 3.0 ± 1.0 0.40 –
0.25 mM Mg2+ dTTP 0.8 0.25 ± 0.09 0.31 ± 0.11 6.1 ± 1.1 0.05 (8) 0.001
6 mM Mg2+ AZTTP 0.4 0.82 ± 0.45 1.5 ± 0.4 4.4 ± 1.8 0.34 –
0.25 mM Mg2+ AZTTP 0.8 0.05 ± 0.03 0.07 ± 0.04 7.2 ± 1.5 0.01 (34) 0.001
6 mM Mg2+ dCTP 1.6 1.2 ± 0.2 0.78 ± 0.15 6.9 ± 2.0 0.11 –
0.25 mM Mg2+ dCTP 1.6 0.42 ± 0.02 0.26 ± 0.01 5.3 ± 2.4 0.05 (2.2) 0.01
6 mM Mg2+ ddCTP 1.6 0.99 ± 0.63 0.62 ± 0.39 5.1 ± 2.3 0.12 –
0.25 mM Mg2+ ddCTP 1.6 0.09 ± 0.07 0.06 ± 0.01 9.7 ± 2.3 0.006 (20) 0.01
6 mM Mg2+ dATP 0.8 1.6 ± 0.3 2.0 ± 0.4 3.5 ± 0.4 0.57 –
0.25 mM Mg2+ dATP 0.8 0.45 ± 0.19 0.56 ± 0.24 5.3 ± 3.1 0.11 (5.2) 0.005
6 mM Mg2+ EFdATP 1.6 1.2 ± 0.4 0.77 ± 0.18 0.97 ± 0.41 0.79 –
0.25 mM Mg2+ EFdATP 1.6 0.29 ± 0.02 0.18 ± 0.01 1.4 ± 0.4 0.13 (6.1) 0.03
a

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, a C opposite a G, or an A opposite a T for AZTTP, ddCTP, or EFdATP, respectively. All assays used one of the following RT concentrations: 0.4, 0.8, or 1.6 nM. Results are averages of three experiments ± SD.

b

kcat was calculated by dividing Vmax by the enzyme concentration.

c

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

d

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