title: NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.
abstract: The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general increase in the Hill coefficients observed in the complex is consistent with the screening of the interacting lysine residues by the DNA. The pKa of K312 residue increased to 10.58 in the complex, probably due to salt bridge formation with the 5'-phosphate group of the DNA. The pKa values obtained for the lyase domain of Pol lambda in the present study are consistent with recent crystallographic studies of Pol beta complexed with 5-phosphorylated abasic sugar analogues in nicked DNA which reveal an open site with no obvious interactions that would significantly depress the pK value for the active site lysine residue. It is suggested that due to the heterogeneity of the damaged DNA substrates with which Pol lambda as well as other related polymerases may be required to bind, the unexpectedly poor optimization of the lyase catalytic site may reflect a compromise of flexibility with catalytic efficiency.
authors.affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Guanghua
authors.initials: G
authors.lastname: Gao
authors.forename: Eugene F
authors.initials: EF
authors.lastname: DeRose
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Kirby
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005663:Furans / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry
chemical_list: D005663:Furans; D011522:Protons; D012545:Schiff Bases; C018674:tetrahydrofuran; C423906:5'-deoxyribose phosphate lyase; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta; D019761:Phosphorus-Oxygen Lyases; D008239:Lysine