Table 1.

Kinetic parameters of Kemp eliminases.

Enzyme	Mutations from HG3a	kcat/KM (M−1 s−1)b
HG3	–	146 ± 6 (1300)
HG3.3b	V6I K50H M84C S89R Q90D A125N	2200 ± 100 (5400)
HG3.7	V6I Q37K K50Q M84C S89R Q90H A125N	27,000 ± 2000 (37,000)
HG3.14	V6I Q37K K50Q G82A M84C Q90H T105I A125T T142N T208M T279S D300N	52,000 ± 1000 (70,000)
HG3.17	V6I Q37K N47E K50Q G82A M84C S89N Q90F T105I A125T T142N T208M F267M W275A R276F T279S D300N	126,000 ± 9000 (230,000)
HG4	K50Q G82A M84C Q90F A125T F267M W275A R276F	103,000 ± 4000

^aMutations in italics occurred at sites optimized during the computational design of HG2⁵.

^bIndividual parameters K_M and k_cat could not be determined accurately because saturation was not possible at the maximum substrate concentration tested (2 mM), which is the substrate’s solubility limit (Supplementary Fig. 2). Catalytic efficiencies (k_cat/K_M) were calculated from the slope of the linear portion ([S] ≪ K_M) of the Michaelis-Menten model (v₀ = (k_cat/K_M)[E₀][S]). n = 2 independent experiments for HG3, HG3.3b, HG3.7, and HG3.17. n = 3 independent experiments for HG3.14 and HG4. Errors of linear regression fitting, which represent the absolute measure of the typical distance that each data point falls from the regression line, are provided. Values in parentheses are from Blomberg et al.¹⁵.