Table 2. A comparison of calculated and experimental activation free energies for the Kemp elimination of 5-nitrobenzisoxazole by the GNCA4-WT β-lactamase and a series of active site mutants ^a .

Variant	k cat	K m	k cat/Km	ΔG‡exp	ΔG‡calc
GNCA4-WT (no His-tag)	2.6 ± 0.44	1.5 ± 0.4	1705 ± 139	16.7	16.2 ± 0.1
G62S	3.64 ± 0.83	1.25 ± 0.45	2911 ± 401	16.7	16.3 ± 0.2
A146G	5.44 ± 0.77	2.34 ± 0.44	2328 ± 112	16.5	16.5 ± 0.2
A173V	3.78 ± 0.19	1.53 ± 0.12	2464 ± 62	16.7	16.9 ± 0.3
L265Q	4.4 ± 1.01	1.8 ± 0.58	2447 ± 242	16.6	16.7 ± 0.2
R256K	6.13 ± 1.76	3.2 ± 1.1	1542 ± 369	16.4	16.9 ± 0.2
R256A	4.80 ± 1.40	4.7 ± 1.6	875 ± 15	16.5	16.6 ± 0.3

^aThe GNCA4-WT β-lactamase, which is used as the baseline for our study, is referred to in this table as “wild-type” (“GNCA4-WT”). Note that this data for the “wild type” was measured without a His-tag in ref. 35, which accounts for the small difference with the data given in Table 1 (taken also from ref. 35). Kinetic measurements were performed as described in the Methodology section, and k_cat, K_M, and k_cat/K_M values are provided in s^–1, mM, and M^–1 s^–1, respectively. ΔG‡exp and ΔG‡calc denote the experimental and calculated activation free energies for these enzymes, in kcal mol^–1. ΔG‡exp was derived from k_cat using transition state theory, and ΔG‡calc is shown as averages and standard error of the mean over thirty individual EVB trajectories per system. All the values in this table were measured at pH 7 with no acetonitrile (other than the small amount coming from the substrate stock solution). All kinetic measurements were performed at 25 °C.