pKa 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 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.
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Department of Molecular Biology and Biochemistry, University of California-Irvine, Irvine, California 92697-3900, USA.
Gershon
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D000595:Amino Acid Sequence; D002244:Carbon / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D015335:Molecular Probes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011497:Protein O-Methyltransferase / Q000737:chemistry* / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D013379:Substrate Specificity
D015335:Molecular Probes; D012315:RNA Caps; D002244:Carbon; D011497:Protein O-Methyltransferase; D008239:Lysine