Table 2. Summary of pH-independent P_i dissociation constants for AP active site nucleophile mutants.

Residue 102	Residue 166	(M)a	(M)b	(M)c	(M)d	(M)e
Ser	Arg	4.6×10−7	≥1×10−7	≤2.9×10−13	—	—
Gly	Arg	—	—	—	(∼1×10 −15 )	(∼1×10 −8 )
Ser	Ser	1.1×10−4	≥2.5×10−6	≤6.9×10−11	—	—
Gly	Ser	—	—	—	2.1×10−13	9.3×10−8

—, not applicable.

^aDissociation constant for binding to deprotonated Ser102 as defined in Figure 4C from the measured pH-dependent P_i affinity in Figure 4A for R166S AP and Figure S9C for WT AP. Note that binding of results in a proton transfer to the enzyme as illustrated in Equation 1; represents the observed overall binding.

^bLower limit of the dissociation constant for binding to deprotonated Ser102 AP determined as described in Text S6 and Figure S9.

^cUpper limit for the dissociation constant for binding to protonated Ser102 AP as described in the main text and Figure 5.

^dDissociation constant for binding to S102G AP estimated from the measured affinity of S102G/R166S AP and the expected contribution of Arg166 of 240-fold to the affinity (cf., for WT and R166S AP). The dissociation constant for binding to S102G/R166S AP was determined from the pH-dependent binding data in Figure 4A and is defined in the model in Figure 4D.

^eDissociation constant for binding to S102G AP estimated based on an expected binding contribution from Arg166 of ∼10-fold (Text S12), and the dissociation constant for binding to S102G/R166S AP determined from the pH-dependent binding in Figure 4A; is defined in the model in Figure 4D.