Table 1.

Characterization of the TEM mutations selected against BLIP

Mutant	Kind	konmut/konwt*	koff† × 10−4 s−1	KD‡ × 10−10 M	Tm,§ °C	Enzymatic activity¶
WT		1.0	0.85	2.8	55	0.0023
T29E	Buried	0.6			47.6	0.0019
K111Q	Interface	0.7			57.6	0.0025
K111M	Interface	0.9			55.7	0.0016
K111N	Interface	0.9	1.5	3.8	54.3	0.0014
S82T	Surface	1.0			53.4	0.0017
P62L	Surface	1.0			53.2	0.0019
H153R	Surface	1.0			58	0.0017
V108L	Interface	1.0			54.4	0.0015
R240Y	Interface	1.1	1	2.2	55	0.0027
M270L	Interface	1.2			56.4	0.0023
D50L	Surface	1.2			55	0.0015
N100Y	Interface	1.3			52	0.0028
P107Q	Interface	1.3	3.3	6.1	54.3	0.0017
D252K	Surface	1.4			50.8	0.0025
Q99R	Interface	1.6			54.4	0.0019
E168G	Interface	1.6	1	2.1	55.8	0.0014
D252S	Surface	1.6			54.1	0.0012
K111R	Interface	1.9			55.8	0.0012
E110V	Interface	2.3	0.9	0.9	55.5	0.0016
E110G	Interface	3.3	0.5	0.6	56.4	0.0018
E171K	Interface	3.4	0.9	1.0	54	0.001
E110G E168G		3.6	1	1.0	54.6	0.0011
E110V E168G		3.7	0.8	0.8	51	0.0015
E239K	Interface	5.0	1.6	1.4	51.2	0.0019
E239R	Interface	5.2			56	0.0011

^*Stopped-flow measurements as described in Methods. The values are relative to WT (3 × 10⁵ M⁻¹⋅s⁻¹). SE for k_on values is 20%, and for k_onmut/k_onwt is 30% (see Methods for details).

^†From SPR measurements done at six different concentrations. SE is 25%.

^‡K_D = k_off/k_on, with k_off values taken from SPR and k_on from stopped-flow measurements. SE is 30%.

^§Protein thermal stability. SE is 1 °C.

^¶Enzymatic activity was determined as the initial slope of CENTA catalysis using 5 nM enzyme and 375 µM substrate. SE is 30%.