title: Determination of lysine pK values using [5-13C]lysine: application to the lyase domain of DNA Pol beta.
abstract: Determination of the protonation state of titratable protein residues is of critical importance for the interpretation of active site chemistry, as well as for understanding the role of electrostatic interactions in protein folding and stability. However, protein titration studies are limited by the fact that, at extreme pH values, increasing fractions of unfolded or partially unfolded structures may be present. This problem is particularly acute for lysine residues which have high pK values. In the present study, we point out that the use of the 13C resonance of lysine C-5 as a reporter for titration of the epsilon-amino group is preferable to the use of C-6 due to the 5-fold greater titration shift, so that reasonable results can be obtained using a two parameter fit of data obtained over a more limited pH range. A new synthetic procedure for [5-13C]lysine is described, and the pK value for Lys72 in the lyase domain of DNA polymerase beta has been determined using the [5-13C]lysine-labeled enzyme. The results agree well with recent studies of the Pol lambda lyase domain, demonstrating that the pK value for this residue is not optimized for Schiff base chemistry (Gao et al., Biochemistry 2006, 45, 1785-1794). We also have re-evaluated data for the pK of Lys73 in the TEM-1 beta-lactamase.
authors.affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Guanghua
authors.initials: G
authors.lastname: Gao
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
authors.forename: Siegfried N
authors.initials: SN
authors.lastname: Lodwig
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Unkefer
authors.forename: William A
authors.initials: WA
authors.lastname: Beard
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
mesh_terms: D001665:Binding Sites; D002247:Carbon Isotopes; D020134:Catalytic Domain; D019951:DNA Polymerase beta / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary
chemical_list: D002247:Carbon Isotopes; D019951:DNA Polymerase beta; D008239:Lysine