Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole
Figure 9
Binding Assay for Phenolates via Competition with a Fluorescently Labeled Equilenin (EqA488–1)
(A) Structure of EqA488. Synthesis resulted in two isomers, and the isomer referred to as EqA488-1 was used in further experiments, as described in Materials and Methods.
(B) Addition of pKSI D40N (E) to 0.1 nM EqA488–1 (Eq) (pH 6.9) leads to quenching of fluorescence at 515 nm (excitation at 480 nm). Each point is the average of two replicates (with errors smaller than the points). Data were fit to Equation 1 and gave = 0.7 ± 0.1 nM for this determination. Additional replicates gave = 1.0 ± 0.3 nM. The accuracy of this determination does not impact the comparison of the affinities of the substituted phenolates relative to one another.
(C) Addition of 4-nitrophenol (P) to a solution of 0.1 nM EqA488 and 5 nM pKSI D40N pH 6.9 leads to recovery of fluorescence. Data were fit to Equation 2 and gave = 11.0 ± 0.9 μM. This observed affinity was first converted to an apparent affinity (= 1.8 ± 0.2 μM; Equation 3) of the phenol for the enzyme at (pH 6.9). This apparent affinity was then converted into the pH-independent affinity ( = 26 ± 2 nM; Equation 4) of the phenolate form of the ligand (PO−) for the protonated form of the enzyme (EOH) using the known phenol and enzyme ionization constants ( = 7.1 and = 5.5, respectively; see Materials and Methods).
(D) Binding schemes from which Equations 1–4 were derived.