Table 2.

pK_a values and shifts of the predicted general acid (C10:N3) and nucleophile (A63:N1) nucleotides in the ligand-bound form of MTR1^a

		Soln.	MTR1 (bound)		Expt.
Nucleotide	Role	pKa	ΔpKa	pKa	App. pKa
C10	General acid	4.2	2.06(03)	6.26	6.2
A63	Nucleophile	3.5	−0.74(62)	2.76	5.0

^aShifts are calculated relative to experimental solution values (55,56) (Soln.) from AFE simulations for MTR1 bound to the O⁶mG substrate. The experimental apparent pK_a values are shown for reference; however, the respective roles were not experimentally validated. Values shown in bold are parameters used to construct the activity-pH profiles in Figure 4. Analysis of enhanced sampling for AFE simulations used to determine the pK_a shifts is provided in Supplementary Table S5 and Supplementary Figure S8.