Table 1. Calculated Activation (ΔG^‡) and Reaction Free Energies (ΔG⁰), Obtained Using the Empirical Valence Bond Approach, As Well As Relevant Corresponding Experimental Observables, For the Decarboxylation of Compounds 1a through 1e by Wild-Type AMDase and Variants^a.

system		Pro-(R)		Pro-(S)		experimental data
		ΔG‡	ΔG0	ΔG‡	ΔG0	selectivity	kcat	ΔG‡exp
1a	WT	15.6 ± 0.4	14.0 ± 0.6	26.6 ± 0.6	24.9 ± 0.6	(R)	27923	14.123
	G74C/C188G	23.1 ± 0.6	21.4 ± 0.6	30.3 ± 0.7	28.7 ± 0.6	(S)	0.00435	21.635
	CLG-IPL	26.8 ± 0.7	24.6 ± 0.7	18.1 ± 0.4	17.2 ± 0.4	(S)	3.835	17.435
1b	WT	15.9 ± 0.7	12.9 ± 0.9	20.2 ± 0.7	18.5 ± 0.8	(R)	15.1,18 3120	16.1,18 15.420
	G74C/C188G	17.9 ± 0.9	14.1 ± 0.9	23.4 ± 0.7	21.7 ± 0.7	(S)
	G74C/C188A	20.7 ± 0.9	17.2 ± 1.0	21.2 ± 0.7	18.0 ± 0.9	(S)
	CLG-IPL	16.7 ± 0.5	14.0 ± 0.7	15.8 ± 0.6	12.5 ± 0.7	(S)	23.7,18 7020	15.9,18 15.020
1c	WT	18.0 ± 0.3	17.1 ± 0.3	24.6 ± 0.9	21.5 ± 1.0	(R)	38.718	15.618
	G74C/C188G	22.7 ± 0.4	20.6 ± 0.5	26.9 ± 0.6	25.3 ± 0.7	(S)	0.07734	19.034
	CLG-IPL	22.3 ± 0.7	20.3 ± 0.7	14.4 ± 0.4	13.7 ± 0.5	(S)	4.318	16.918
1d	WT	28.3 ± 0.8	25.8 ± 0.9	32.9 ± 1.8	29.9 ± 1.7
1e	WT	18.0 ± 0.4	16.3 ± 0.5	35.4 ± 0.7	33.7 ± 0.7	(R)	0.2335	19.135
	CLG-IPL	34.4 ± 1.7	31.7 ± 1.5	17.1 ± 0.6	15.9 ± 0.6	(S)	0.5635	18.635

^aAll calculated values are averages and standard error of the mean over 30 individual EVB trajectories per system, as described in the Methodology section, and shown here are data obtained from modeling the decarboxylation of each compound through cleavage of either the pro-(R) or pro-(S) carboxylate groups. WT denotes the wild-type enzyme. Both experimental and calculated activation and reaction free energies are presented in kcal mol^–1. Shown here are also the experimentally observed selectivities for each compound, as well as the corresponding kinetics (k_cat, s^–1) and activation free energies (ΔG^‡_exp) derived from the experimentally observed activities toward each compound by each variant, as presented in refs (18, 20, 23, 34, and 35). The k_cat values were either taken directly from the literature, or were estimated by using the relationship k_cat = (specific activity × molecular weight). The calculated activation free energies were obtained from the k_cat values using transition state theory at temperature 30 °C (for ref (18)), 37 °C (for ref (35)), and 25 °C for the rest. Note that the specific activities were obtained from bar graphs provided in ref (18) and therefore the experimental kinetics and energetics are only approximate. Blank cells denote that experimental data is not available for a given system.