Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo-exonuclease activity in T5 exonuclease.
T5 5'-3' exonuclease is a member of a homologous group of 5' nucleases which require divalent metal co-factors. Structural and biochemical studies suggest that single-stranded DNA substrates thread through a helical arch or hole in the protein, thus bringing the phosphodiester backbone into close proximity with the active site metal co-factors. In addition to the expected use of Mg(2+), Mn(2+) and Co(2+) as co-factors, we found that divalent zinc, iron, nickel and copper ions also supported catalysis. Such a range of co-factor utilisation is unusual in a single enzyme. Some co-factors such as Mn(2+) stimulated the cleavage of double-stranded closed-circular plasmid DNA. Such endonucleolytic cleavage of circular double-stranded DNA cannot be readily explained by the threading model proposed for the cleavage of substrates with free 5'-ends as the hole observed in the crystal structure of T5 exonuclease is too small to permit the passage of double-stranded DNA. We suggest that such a substrate may gain access to the active site of the enzyme by a process which does not involve threading.
Garforth
S J
SJ
Division of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2RX, UK.
Patel
D
D
Feng
M
M
Sayers
J R
JR
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D017669:Mercury Compounds / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*
D002413:Cations, Divalent; D003067:Coenzymes; D017669:Mercury Compounds; D008670:Metals; D011189:Potassium Chloride; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases