Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor.
The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.
Catalano
C E
CE
Department of Chemistry, Pennsylvania State University, University Park 16802.
Benkovic
S J
SJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors*; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000037:antagonists & inhibitors
D010446:Peptide Fragments; C016205:adenosine 2',3'-riboepoxide 5'-triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V