pK_a of the mRNA Cap-Specific 2‘-O-Methyltransferase Catalytic Lysine by HSQC NMR Detection of a Two-Carbon Probe^†

We have characterized the side chain pK_a for a single lysine analogue within a 316-residue protein containing 21 lysines and 1678 carbon atoms at natural isotope abundance. To do this, the single reactive cysteine of a K175C mutant of VP39 (the mRNA cap-specific 2‘-O-methyltransferase from vaccinia virus) was modified to S-(β-aminoethyl)cysteine (γ-thialysine) using freshly prepared (¹³C)aziridine at room temperature. Modification was accompanied by the rescue of catalytic function at high specific activity. After the fastidious removal of the noncovalently protein-bound aziridine self-polymer using a novel chelating dialysis procedure, signals were monitored by HSQC NMR. Appropriately pH-shifting HSQC NMR peaks were identified in the (¹³C)aziridine-modified enzyme, corresponding to detection of the two covalently attached (¹³C)thioethylamino atoms. The identification was strengthened by comparison with the positions and pH shifts of spectral peaks for tripeptide controls, a small molecule aziridine self-polymer mimetic, and a cysteine-minus control enzyme. pH titration of the modified protein indicated an apparent pK_a of 8.5, consistent with a perturbed pK_a for the catalytic lysine and a model in which the surrounding charged groups direct the lysine ε-amino pK_a via both local electrostatic environment and orbital directionality.