NMR Determination of pK_a Values for Asp, Glu, His, and Lys Mutants at Each Variable Contiguous Enzyme−Inhibitor Contact Position of the Turkey Ovomucoid Third Domain^†,‡

From the larger set of 191 variants at all the variable contact positions in the turkey ovomucoid third domain, we selected a subset that consists of Asp, Glu, His, and Lys residues at eight of the nine contiguous P₆−P_3‘ positions (residues 13−21), the exception being P₃-Cys¹⁶ which is involved in a conserved disulfide bridge. Two-dimensional [¹H,¹H]-TOCSY data were collected for each variant as a function of sample pH. This allowed for the evaluation of 31 of the 32 pK_a values for these residues, the exception being that of P₅-Lys¹⁴, whose signals at high pH could not be resolved from those of other Lys residues in the molecule. Only two of the titrating residues are present in the wild-type protein (P₆-Lys¹³ and P_1‘-Glu¹⁹); hence, these measurements complement earlier measurements by A. D. Robertson and co-workers. This data set was supplemented with results from the pH dependence of NMR spectra of four additional single mutants, P₁-Leu¹⁸Gly, P₁-Leu¹⁸Ala, P₂-Thr¹⁷Val, and P_3‘-Arg²¹Ala, and two double mutants, P₂-Thr¹⁷Val/P_3‘-Arg²¹Ala and P₈-Tyr¹¹Phe/P₆-Lys¹³Asp. Probably the most striking result was observation of a P₂-Thr¹⁷···P_1‘-Glu¹⁹ hydrogen bond and a P_1‘-Glu¹⁹−P_3‘-Arg²¹ electrostatic interaction within the triad of P₂, P_1‘, and P_3‘ (residues 17, 19, and 21, respectively). In several cases, the pK_a of a particular residue was sensed by resonances not only in that residue but also in residue(s) with which it interacts. Remarkably, in several interacting systems, resonances from different protons within the same residue yielded different pH_mid values.