Table 1.

Effect of non-hydrogen-bonding base analogues on Klenow fragment polymerase kinetics

Enzyme	Reaction	Kd(dNTP) (μM)	kpol (s-1)	kpol/Kd (M-1s-1)
Analogues at insertion site:
WT	Template-A + dTTP	11	180	1.6 × 107
WT	Template-A + dFTPa	28	0.52	1.8 × 104
WT	Template-Z + dFTP	68	0.95	1.4 × 104
WT	Template-T + dATP	12	250	2.1 × 107
WT	Template-F + dATP	91	62	6.8 × 105
WT	Template-F + dZTP	88	2.1	2.4 × 104
Klenow fragment mutants in insertion reaction:
E710A	Template-A + dTTPa	17	11	6.2 × 105
E710A	Template-Z + dFTPa	13	0.0067	5.1 × 102
E710A	Template-T + dATPa	6.4	5.6	8.7 × 105
E710A	Template-F + dZTPa	19	0.25	1.4 × 104
Y766A	Template-A + dTTPa	36	20	5.5 × 105
Y766A	Template-Z + dFTPa	26	0.027	1.0 × 103
Y766A	Template-T + dATPa	65	24	3.7 × 105
Y766A	Template-F + dZTP	6.2	0.053	8.6 × 103
F762A	Template-T + dATPb			1.6 × 104
F762A	Template-F + dZTPb			3.2 × 102
Analogues at primer terminus:
WT	(primer)A-T(template) + dGTP	3.0	190	6.3 × 107
WT	(primer)Z-F(template) + dGTP	29	0.0056	1.9 × 102
WT	(primer)Q-F(template) + dGTP	16	0.49	3.1 × 104
WT	(primer)T-A(template) + dGTP	4.5	63	1.4 × 107
WT	(primer)F-Z(template) + dGTP	140	0.16	1.1 × 103
WT	(primer)F-Q(template) + dGTP	81	0.18	2.2 × 103
R668A	(primer)A-T(template) + dGTP	40	2.6	6.6 × 104
R668A	(primer)Z-F(template) + dGTP	37	0.012	3.2 × 102
R668A	(primer)Q-F(template) + dGTP	29	0.008	2.8 × 102

^aSingle measurements. All other determinations were the average of two or more experiments with good agreement.

^bBecause of the high K_d(dNTP) of the F762A mutant protein, it was not possible to use a high enough concentration of dZTP to saturate the reaction, and therefore individual k_pol and K_d values could not be obtained. The efficiency (k_pol/K_d) was determined from the slope of the plot of rate vs. dNTP concentration, at low dNTP concentrations.