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pKa of the mRNA cap-specific 2'-O-methyltransferase catalytic lysine by HSQC NMR detection of a two-carbon probe.

Li C, Gershon PD

Department of Molecular Biology and Biochemistry, University of California-Irvine, Irvine, California 92697-3900, USA.

We have characterized the side chain pKa 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-(beta-aminoethyl)cysteine (gamma-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 pKa of 8.5, consistent with a perturbed pKa for the catalytic lysine and a model in which the surrounding charged groups direct the lysine epsilon-amino pKa via both local electrostatic environment and orbital directionality.

Published 17 January 2006 in Biochemistry, 45(3): 907-17.
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