Novel role of tyrosine in catalysis by human AP endonuclease 1.
Apurinic/apyrimidinic endonuclease (AP endo, HAP1) recognizes abasic sites in ds DNA and makes a single nick in the backbone 5' to the abasic site. In this report we examine the roles of three conserved tyrosine residues in close proximity to the active site. We show that Tyr(128) and Tyr(269), which interact upstream and downstream of the abasic site, respectively, are involved in recognition and binding of abasic site-containing double stranded DNA. However, the two residues are not equivalent, as their effects are differentiated by changes in salt concentration. In sharp contrast, Tyr(171) is directly involved in catalysis as well as binding. Y171F, Y171H, and Y171A all show decreased catalytic efficiencies 25,000-50,000-fold from the WT enzyme. Both imidazole and basic pH markedly stimulate the WT enzyme. Imidazole stimulates Tyr(171) mutant enzymes when tyrosine is also present but basic pH eliminates remaining mutant activity. These results underscore the importance of tyrosines in AP endo catalysis. They render the current hypotheses regarding enzyme action unlikely and allow us to consider the possibility that the phenolate of Tyr(171) is the nucleophile that attacks the scissile phosphate.
Mundle
Sophia T
ST
Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
Fattal
Michael H
MH
Melo
Luisa F
LF
Coriolan
Jean D
JD
O'Regan
N Edel
NE
Strauss
Phyllis R
PR
D019943:Amino Acid Substitution; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent / Q000494:pharmacology; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014443:Tyrosine / Q000737:chemistry*
D002413:Cations, Divalent; D007093:Imidazoles; D014443:Tyrosine; C029899:imidazole; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase