Human DNA polymerase beta deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism.
DNA polymerase beta (beta-pol) cleaves the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5'-incised by AP endonuclease, resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of beta-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of beta-pol are similar. However, kcat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of beta-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5'-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.
Prasad
R
R
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Beard
W A
WA
Strauss
P R
PR
Wilson
S H
SH
D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D019951:DNA Polymerase beta / Q000737:chemistry*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase
D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D004247:DNA; D019951:DNA Polymerase beta; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase