Table S1.

Proteolysis kinetics of GCK variants in the presence of 200 mM glucose

Protein	kop* (x 10−3⋅s−1)	kcl,† s−1	Kop‡ (x 10−2)	ΔG,§ kcal/mol
Wild-type	1.0 ± 0.1	0.34 ± 0.12	0.29 ± 0.09	3.46 ± 0.3
α13-helix + β-hairpin	0.03 ± 0.0003	0.025 ± 0.002	0.12 ± 0.01	4.00 ± 0.4
α-type activation
S64P	0.12 ± 0.009	0.22 ± 0.05	0.056 ± 0.01	3.99 ± 0.1
T65I	0.018 ± 0.0007	1.2 ± 0.22	0.0015 ± 0.0003	6.58 ± 1.2
V455M	0.16 ± 0.01	0.37 ± 0.08	0.043 ± 0.009	4.59 ± 0.2
α13 helix	0.0089 ± 0.0002	0.82 ± 0.08	0.0011 ± 0.0009	6.77 ± 0.08
β-type activation
M197V	0.53 ± 0.04	0.77 ± 0.15	0.070 ± 0.01	4.31 ± 0.2
I211F	0.15 ± 0.02	0.28 ± 0.08	0.056 ± 0.02	4.43 ± 0.2
Y214C	6.2 ± 0.4	0.73 ± 0.13	0.86 ± 0.1	2.82 ± 0.1
β-hairpin	4.0 ± 1.0	0.91 ± 0.45	0.44 ± 0.19	3.22 ± 1.4

^*Rate constant for opening to the cleavable state.

^†Rate constant for closing from the cleavable state.

^‡Equilibrium constant between the cleavable and uncleavable states.

^§Free energy difference between the cleavable and uncleavable states.