Table 1. Calculated and Experimental Activation Barriers (ΔG^⧧), and Changes in Gibbs Free Energy (ΔG₀) for Hydride Transfer from NADH to DHAP, Catalyzed by Wild-Type and Variants of Glycerol-3-phosphate Dehydrogenase (hlGPDH) at 25 °C^a.

variant	ΔGexp⧧	ΔGcalc⧧	ΔG0,calc	ΔΔGWT→mut,exp⧧b	ΔΔGWT→mut,calc⧧c	ΔΔGcalc→exp⧧d
wild-type	14.2	14.2 ± 0.3	–6.2 ± 0.3	0.0	0.0	0.0
K120A	17.5	21.1 ± 0.2	0.4 ± 0.4	3.3	6.9	3.6
K120A/D260[H+]e	17.5	15.0 ± 0.2	–8.4 ± 0.3	3.3	0.8	–2.5
R269A	20.5	16.7 ± 0.2	–1.1 ± 0.3	6.3	2.5	–3.8
Q295A	14.7	15.6 ± 0.3	–4.9 ± 0.4	0.5	1.4	0.9

^aThe calculated energies are averages and SEM over 30 independent trajectories per system, determined as described in the Methodology section.

^bThe difference between the experimental activation barrier for the hydride transfer reaction catalyzed by wild-type hlGPDH and the variant enzyme (ΔΔG_WT→mut,exp^⧧).

^cThe difference between the calculated activation free energies for the hydride transfer reaction catalyzed by wild-type hlGPDH and the variant enzyme (ΔΔG_{WT→mut,calc}^⧧).

^dThe difference between the experimental and calculated activation free energies for the hydride transfer reaction catalyzed by wild-type hlGPDH or the enzyme variant (ΔΔG_calc→exp^⧧).

^eThe EVB calculated energetics for the K120A variant with the D260 side chain protonated include a 2 kcal·mol^–1 correction for the protonation of this side chain, based on a calculated pK_a of 6 from PROPKA 3.1,³⁴ obtained as described in the main text. The experimental activation free energies are calculated from kinetic data presented in refs (5, 20, 66) and in Table S5. All energies are presented in kcal·mol^–1.